Abstract:
A method of analyzing a biological specimen to detect cancer in a subject, involving determining an ultraconserved RNA in the specimen and comparing the expression level to a control. The ultraconseved RNA may be uc.338 and may be used to detect hepatocellular cancer. Transcript RNA encoding ultraconserved RNA may also be used to detect cancer. Anti-cancer compositions and tumor-suppressing agents for treating cancer are also provided.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims priority of U.S. provisional application Ser. No. 61/322,608, filed Apr. 9, 2010, the disclosure of which is incorporated herein by reference as if fully recited herein. 
     
    
     TECHNICAL FIELD 
       [0002]    Embodiments relate to methods, compositions, and systems for detecting and treating cancer in a subject, particularly to method&#39;s, compositions, and systems for detecting and regulating ultraconserved RNA or transcript RNA. 
       BACKGROUND 
       [0003]    Hepatocellular carcinogenesis involves a complex interaction of genes resulting in variable modulation of key pathways involved in tumor cell growth. Using molecular techniques for global genomic profiling, the transcriptome in hepatocellular cancers (HCC) has been described, and several genes that are differentially activated have been identified. The major focus of attention in these efforts has been on the characterization of expression of protein-coding genes and their use for determining clinical outcomes. However, the majority of the human genome consists of non-protein-coding RNA (ncRNA), some of which is transcribed. Increasing evidence points to an important functional or regulatory role of ncRNA in cellular processes, as well as a contribution of aberrant ncRNA expression to disease phenotypes. 
         [0004]    Along with the highly abundant transfer and ribosomal RNAs, ncRNAs include microRNAs that modulate mRNA expression, small nucleolar RNAs that guide chemical modification of RNA-molecules, small interfering RNAs (siRNAs) that account for the interference pathway, piwiRNAs that are linked to transcriptional gene silencing of retrotransposons and long non coding RNAs whose role is still unknown. The role played by the non-coding RNA genome in malignant transformation and tumor growth in HCC is being increasingly recognized. Researchers have recently provided data from profiling studies in which several microRNAs were identified and shown to be involved in the modulation of cell proliferation and apoptosis. Recent studies revealing the presence of several hundred long transcribed ncRNAs raise the possibility that many ncRNAs contributing to cancer remain to be discovered. Other than microRNAs, however, only a handful of ncRNA have been implicated in hepatocarcinogenesis. For the most part, the function of these ncRNAs is unknown. Sequence conservation across species has been postulated to indicate that a given ncRNA may have a cellular function. A genome-wide survey identified several hundred ncRNAs with a size greater than 200 bp that showed a remarkable conservation with 100% identity across the human, mouse and rat genomes. These highly conserved long ncRNAs have been named ultraconserved regions, and are conserved across many other species as well, with 99% of these genes showing high levels of conservation within the dog, 97% within the chicken and 67% within the fugu genomes. Their wide distribution in the genome and lack of natural variation in the human population suggested that these ncRNA genes have a biological function which is essential for normal cells. However, the function of these ultraconserved genes remains controversial and unknown. 
       SUMMARY 
       [0005]    Embodiments relate to a method of analyzing a biological specimen to detect cancer in a subject, comprising the steps of: (a) determining the expression level of a RNA sequence in the specimen; and (b) comparing the expression level to a control, wherein a pre-identified difference between the determined expression level and the control is indicative of cancer in the subject. In some embodiments, the RNA sequence is uc.338. In various embodiments, the RNA sequence is selected from the groups consisting of uc.338, uc.24, uc.189, uc.134, uc.378, uc.349, uc.78, uc.233, uc.262, uc.331, uc.136, and uc.246. In various other embodiments, the RNA sequence is selected from the groups consisting of uc.110, uc.473, uc.275, uc.477, uc.269, uc.448, and uc.20. In some embodiments, the RNA sequence may be TUC338 or any other transcript RNA that is capable of encoding an ultraconserved RNA selected from the group consisting of uc.338, uc.24, uc.189, uc.134, uc.378, uc.349, uc.78, uc.233, uc.262, uc.331, uc.136, and uc.246. In various other embodiments, the RNA sequence is one that is capable of encoding an ultraconserved RNA selected from the group consisting of uc.110, uc.473, uc.275, uc.477, uc.269, uc.448, and uc.20. 
         [0006]    In some embodiments, the subject is a mammal. In specific embodiments, the subject is a human. 
         [0007]    In exemplary embodiments, the cancer is a hepatocellular cancer. In specific embodiments, the subject is suspected of having HCC or is at risk for HCC. In some embodiments, the control is a non-malignant hepatocyte. 
         [0008]    In some embodiments, the expression of uc.338 is determined by an amplification assay or a hybridization assay. 
         [0009]    Embodiments are also directed towards an isolated small inhibitory ribonucleic acid (“siRNA”) molecule that inhibits expression of a nucleic acid molecule encoding an ultraconserved RNA molecule. In various embodiments, the ultraconserved RNA molecule is selected from the group consisting of uc.338, uc.24, uc.189, uc.134, uc.378, uc.349, uc.78, uc.233, uc.262, uc.331, uc.136, and uc.246. In other embodiments, the ultraconserved RNA is selected from the group consisting of uc.110, uc.473, uc.275, uc.477, uc.269, uc.448, and uc.20. In embodiments, the ultraconserved RNA is uc.338. In various embodiments, the siRNA comprises an isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO: 6. In other embodiments, the siRNA comprises an isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO: 7. 
         [0010]    Various embodiments are directed towards an anti-cancer composition comprising an antisense oligonucleotide, ribozyme, siRNA, or any combination thereof, that binds to uc.338. 
         [0011]    Other embodiments are directed to a tumor-suppressing agent comprising as an active ingredient, at least one of: a double stranded RNA complementary to a transcript of an ultraconserved RNA gene; a DNA encoding a double-stranded RNA complementary to a transcript of an ultraconserved RNA gene; and a vector carrying, as an insert, a DNA encoding a double-stranded RNA complementary to a transcript of an ultraconserved RNA gene. In various embodiments, the ultraconserved RNA gene is selected from the group consisting of uc.338, uc.24, uc.189, uc.134, uc.378, uc.349, uc.78, uc.233, uc.262, uc.331, uc.136, and uc.246. In some embodiments, the ultraconserved RNA gene is selected from the group consisting of uc.110, uc.473, uc.275, uc.477, uc.269, uc.448, and uc.20. In various embodiments, the ultraconserved RNA is uc.338. In various embodiments the transcript is TUC338. 
       DESCRIPTION OF THE SEQUENCES 
       [0012]    The various embodiments can be more fully understood from the following detailed description and the accompanying sequence descriptions, which form a part of this application. 
         [0000]    
       
         
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 SEQ 
                   
                   
               
               
                 ID 
                   
                   
               
               
                 NO: 
                 Name 
                 Sequence 
               
               
                   
               
             
             
               
                 1 
                 uc.338 (DNA) 
                 TGACAAGGTGCCGGATTGACAGCCCTGGAG 
               
               
                   
                   
                 ACTGAAATCCTCTATTTATCCACAGGACAGGT 
               
               
                   
                   
                 ACAGCACAGGCAGCGACAGTGCGAGCTTTC 
               
               
                   
                   
                 CCCACACCACCCCGTCCATGTGCCTCAACCC 
               
               
                   
                   
                 TGACCTGGAGGGACCACCTCTAGAGGTGAG 
               
               
                   
                   
                 AGGGGATGTTCAGTCTCCAAGGCTCACTCAA 
               
               
                   
                   
                 TCCTTCCGCCTCAGCCGAGACTGCCAACACA 
               
               
                   
                   
                 CGGGGGGC 
               
               
                   
               
               
                 2 
                 uc.338(for 
                 AGCGACAGTGCGAGCTTT 
               
               
                   
                 RTpcr): 
                   
               
               
                   
               
               
                 3 
                 uc.338(for 
                 GGAAGGATTGAGTGAGCCTT 
               
               
                   
                 RTpcr): 
                   
               
               
                   
               
               
                 4 
                 uc.338-F: 
                 TTTTGTGTGAATTTCATGCTGGT 
               
               
                   
               
               
                 5 
                 uc.338-R: 
                 GGTGTCCACAGCACCAAAAA 
               
               
                   
               
               
                 6 
                 siRNA-1 (RNA) 
                 UGACAGCCCUGGAGACUGA 
               
               
                   
               
               
                 7 
                 siRNA-2 (RNA) 
                 CCACAGGACAGGUACAGCA 
               
               
                   
               
               
                 8 
                 TUC338 
                 AAGCAGTGGTATCAACGCAGAGTACATGGGAG 
               
               
                   
                   
                 TGGGCTCTGTAGGGGTTCAACTTAATCCCACA 
               
               
                   
                   
                 CTGACCAGGAGCTGGCATCAGCTCTGTTGCCC 
               
               
                   
                   
                 CACCCCTTCATTCTTATTGAAAATAGCCCAAT 
               
               
                   
                   
                 TGTGCAGTGTGAAATCTGCCACTAGGTAACTT 
               
               
                   
                   
                 TTTTTTTTAATTCACTGATTACAGCTCGTTTC 
               
               
                   
                   
                 AAATTGTGTCTGAGCTCCTTTTTAAAGGAAAA 
               
               
                   
                   
                 AGGAAAAAAATAAACAACTTTTTTTGTGTGAA 
               
               
                   
                   
                 TTTCATGCTGGTGACAAGGTGCCGGATTGACA 
               
               
                   
                   
                 GCCCTGGAGAGACTGAAATCCTCTATTTATCC 
               
               
                   
                   
                 ACAGGACAGGTACAGCACAGGCAGCGACAGTG 
               
               
                   
                   
                 CGAGCTTTCCCCACACCACCCCGTCCATGTGC 
               
               
                   
                   
                 CTCAACCCTGACCTGGAGGGACCACCTCTAGA 
               
               
                   
                   
                 GGTGAGAGGGGATGTTCAGTCTCCAAGGCTCA 
               
               
                   
                   
                 CTCAATCCTTCCGCCTCAGCCGAGACTGCCAA 
               
               
                   
                   
                 CACACGGGGGGCCAGTGGCGCTGGTGATTTTT 
               
               
                   
                   
                 GGTGCTGTGGACACCACCTGTCCACGGGGACC 
               
               
                   
                   
                 TGGACTGACCCCCCCAACCTCATTTCACCCAG 
               
               
                   
                   
                 GCCGCGTAGCCCACCAGATGGTAACACCAACT 
               
               
                   
                   
                 TTTTTTTTTTTTTT 
               
               
                   
               
             
          
         
       
     
         [0013]    SEQ ID NOS: 2-5 are examples of primers and primer sequences that can be used for PCR amplification. However, it is appreciated that various alternative primers may be designed. 
         [0014]    Other features and advantages will be apparent from the following detailed description, and from the claims. 
     
    
     
       DRAWINGS 
         [0015]    A better understanding of the embodiments will be obtained from a reading of the following detailed description and the accompanying drawings in which: 
           [0016]      FIG. 1 . ucRNAs are aberrantly expressed in malignant hepatocytes. Panel A: Genome-wide expression profiling was performed using a custom microarray in HepG2 cells and normal human hepatocytes (HH). 56 ucRNAs were aberrantly expressed in malignant hepatocytes with a p value&lt;0.05, with twelve ucRNAs increased and 7 decreased by greater than 2-fold as shown in Table 2 below: 
           [0000]    
         
           
                 
                 
                 
                 
               
                 
                 
                 
                 
               
             
                 
                   TABLE 2 
                 
                 
                     
                 
                 
                     
                   FOLD CHANGE 
                   FOLD CHANGE, 
                   Parametric 
                 
                 
                   ucRNA 
                   HCC vs HH 
                   log2 
                   p-value 
                 
                 
                     
                 
               
               
                 
                     
                 
               
            
             
                 
                   uc.338+ 
                   6.99 
                   2.81 
                   0.0001277 
                 
                 
                   uc.24 + A 
                   4.75 
                   2.25 
                   0.0466492 
                 
                 
                   uc.189+ 
                   4.23 
                   2.08 
                   0.0000211 
                 
                 
                   uc.134 + A 
                   3.78 
                   1.92 
                   0.000059 
                 
                 
                   uc.378+ 
                   2.98 
                   1.57 
                   0.0000213 
                 
                 
                   uc.349+ 
                   2.96 
                   1.57 
                   0.0001016 
                 
                 
                   uc.78+ 
                   2.69 
                   1.43 
                   0.0001255 
                 
                 
                   uc.233 + A 
                   2.37 
                   1.24 
                   0.0008148 
                 
                 
                   uc.262 + A 
                   2.30 
                   1.20 
                   0.0056489 
                 
                 
                   uc.331+ 
                   2.24 
                   1.17 
                   0.0000605 
                 
                 
                   uc.136+ 
                   2.18 
                   1.12 
                   0.0020847 
                 
                 
                   uc.246+ 
                   2.00 
                   1.00 
                   0.0001246 
                 
                 
                   uc.47+ 
                   1.96 
                   0.97 
                   0.008174 
                 
                 
                   uc.204 + A 
                   1.94 
                   0.96 
                   0.0225475 
                 
                 
                   uc.139+ 
                   1.92 
                   0.94 
                   0.0406478 
                 
                 
                   uc.362 + A 
                   1.88 
                   0.91 
                   0.0108186 
                 
                 
                   uc.10 + A 
                   1.86 
                   0.90 
                   0.0021238 
                 
                 
                   uc.339+ 
                   1.82 
                   0.86 
                   0.0007036 
                 
                 
                   uc.420+ 
                   1.76 
                   0.81 
                   0.0011707 
                 
                 
                   uc.412 + A 
                   1.75 
                   0.81 
                   0.0070073 
                 
                 
                   uc.234+ 
                   1.74 
                   0.80 
                   0.0024131 
                 
                 
                   uc.372+ 
                   1.70 
                   0.77 
                   0.0379089 
                 
                 
                   uc.44 + A 
                   1.60 
                   0.67 
                   0.0048444 
                 
                 
                   uc.456 + A 
                   1.54 
                   0.62 
                   0.0114 
                 
                 
                   uc.190 + A 
                   1.53 
                   0.61 
                   0.0081303 
                 
                 
                   uc.177 + A 
                   1.51 
                   0.59 
                   0.0115514 
                 
                 
                   uc.392 + A 
                   1.49 
                   0.57 
                   0.0195016 
                 
                 
                   uc.278+ 
                   1.46 
                   0.54 
                   0.041165 
                 
                 
                   uc.470 + A 
                   1.44 
                   0.53 
                   0.0081265 
                 
                 
                   uc.213+ 
                   1.41 
                   0.50 
                   0.0249108 
                 
                 
                   uc.117 + A 
                   1.36 
                   0.44 
                   0.0153249 
                 
                 
                   uc.1+ 
                   1.31 
                   0.39 
                   0.0414189 
                 
                 
                   uc.346+ 
                   1.31 
                   0.39 
                   0.0463969 
                 
                 
                   uc.475+ 
                   −1.24 
                   −0.31 
                   0.0418998 
                 
                 
                   uc.95+ 
                   −1.30 
                   −0.38 
                   0.0363832 
                 
                 
                   uc.462+ 
                   −1.32 
                   −0.40 
                   0.0183523 
                 
                 
                   uc.16+ 
                   −1.38 
                   −0.47 
                   0.0491905 
                 
                 
                   uc.369+ 
                   −1.40 
                   −0.49 
                   0.0473564 
                 
                 
                   uc.100+ 
                   −1.42 
                   −0.51 
                   0.0076957 
                 
                 
                   uc.48+ 
                   −1.47 
                   −0.55 
                   0.0418279 
                 
                 
                   uc.305+ 
                   −1.55 
                   −0.63 
                   0.0114024 
                 
                 
                   uc.88+ 
                   −1.63 
                   −0.70 
                   0.0015275 
                 
                 
                   uc.325 + A 
                   −1.66 
                   −0.73 
                   0.0068587 
                 
                 
                   uc.388+ 
                   −1.66 
                   −0.73 
                   0.0018223 
                 
                 
                   uc.153+ 
                   −1.79 
                   −0.84 
                   0.00126 
                 
                 
                   uc.411 + A 
                   −1.87 
                   −0.91 
                   0.0059916 
                 
                 
                   uc.18+ 
                   −1.88 
                   −0.91 
                   0.0105249 
                 
                 
                   uc.128 + A 
                   −1.89 
                   −0.92 
                   0.0009112 
                 
                 
                   uc.33 + A 
                   −1.92 
                   −0.94 
                   0.011849 
                 
                 
                   uc.110 + A 
                   −2.06 
                   −1.04 
                   0.000097 
                 
                 
                   uc.473+ 
                   −2.12 
                   −1.09 
                   0.0000668 
                 
                 
                   uc.275+ 
                   −2.23 
                   −1.16 
                   0.0001138 
                 
                 
                   uc.477+ 
                   −2.44 
                   −1.29 
                   0.0001645 
                 
                 
                   uc.269 + A 
                   −2.71 
                   −1.44 
                   0.0008003 
                 
                 
                   uc.448+ 
                   −3.15 
                   −1.66 
                   0.0000296 
                 
                 
                   uc.20 + A 
                   −3.42 
                   −1.78 
                   0.0000035 
                 
                 
                     
                 
               
            
           
         
       
           [0017]    The ratio of expression of these ucRNAs in malignant HepG2 cells relative to HH is plotted against the p-value. Selected ucRNAs with a greater than 3-fold change in expression are annotated. Panel B: The genomic locations of the ucRNA as exonic, non exonic or possibly exonic relative to protein-coding genes is depicted for all ucRNAs and for the group of ucRNAs that are aberrantly expressed in malignant hepatocytes. Selective enrichment of a specific group of ucRNA based on relationship to known protein-coding genes was not observed. 
           [0018]      FIG. 2 . uc.338 is overexpressed in HCC cells lines. Panel A: Schematic representation of the partial exonic location of uc.338 within the PCBP2 gene. The exons of PCBP2 are indicated by dark grey boxes, while uc.338 is depicted as the light grey box. The location of the uc.338 forward (F) and reverse (R) primers used for real-time-PCR and probe used for in situ hybridization are shown. With the exception of the forward primer, these are located within the PCBP2 intronic region. Of the siRNAs targeting uc.338, siRNA-1 is entirely intronic while siRNA-2 overlaps a few nucleotides of the coding sequence of PCBP2. F′ and R′ indicate primers used for detection of PCBP2. Panel B: RNA was extracted from different cell lines and uc.338 expression evaluated by quantitative real-time-PCR. The expression of uc.338 was normalized to that of RNU6. Bars represent the mean and SEM of 4 replicates. uc.338 expression was increased in all HCC cell lines compared to normal human hepatocytes (HH). * p&lt;0.05 relative to HH. Non-malignant epithelial cells (hatched bars): HE: hepatocytes, BE: biliary epithelia, PE: prostatic epithelia. Malignant cells (solid bars): HCC: hepatocellular cancer, CCA: cholangiocarcinoma, PaC: Pancreatic cancer, CRC: colorectal cancer, PC: prostate cancer, BC: breast cancer. 
           [0019]      FIG. 3 . Representation of the different classes of staining after in situ hybridization with locked nucleic acid (LNA)-anit-uc.338. 
           [0020]      FIG. 4 . uc.338 is over-expressed in human HCC tissues. uc.338 expression was evaluated in a total of 221 HCCs, 72 cases of non cirrhotic and 97 cases of cirrhotic adjacent liver tissues. Paraffin-embedded, formalin-fixed liver tissues were incubated with LNA-anti-uc.338. Panel A: uc.338 expression was classified as negative, weak, moderate, or strong based on the percentage of cells with detectable staining for uc.338. The proportion of cases of HCC, cirrhotic liver, or non-cirrhotic liver within each class is depicted in the columns. Panel B: The mean and 95% confidence intervals of uc.338 expression in non-cirrhotic liver, cirrhotic liver and HCC tissues is shown. *p&lt;0.05. Panel C: uc.338 expression was compared between 156 HCCs and their corresponding adjacent liver tissues. Picture of representative cases are shown. Panel D: An expression score was derived as the ratio of the difference in expression between HCC and adjacent liver tissues to the standard deviation of uc338 in all tissues, and plotted with the size of the bubble representing the number of cases. 
           [0021]      FIG. 5 . Nuclear expression of uc.338 in HCC cells. Panel A: Paraffin-embedded, formalin-fixed liver tissues were incubated with LNA probe anti-uc.338. uc.338 was frequently detected in nuclei of HCC in situ. Panel B: RNA was extracted from the nuclear and the cytoplasmic fraction of HCC cells and uc.338 expression evaluated by real-time-PCR. Bars represent the mean and SEM of relative expression of uc.338 from two experiments performed in four replicates. * p&lt;0.05. 
           [0022]      FIG. 6 . uc.338 and PCBP2 are independently regulated. Panel A: Expression of uc.338 is plotted against PCBP2 mRNA expression in samples of normal and HCC cell lines. There is no linear correlation between uc.338 and PCBP2 gene expression (P=0.08). Panel B: HepG2 cells were transfected with siRNA against PCBP2 or siRNA control. Bar represents the mean of 2 independent experiments performed in four replicates. *p&lt;0.05 compared to control siRNA. Panel C: The expression of uc.338 was decreased in HepG2 and Huh-7 cells using two different siRNAs against uc.338. After 48 hours RNA was collected and uc.338 and PCBP2 expression evaluated by real time PCR. Bars represent the mean and SEM of 3 experiments performed in 4 replicates. *p&lt;0.05 relative to siRNA control. These data indicate that uc.338 does not regulate the expression of PCBP2 and that the expression of uc.338 is independent of PCBP2. 
           [0023]      FIG. 7 . Schematic Representation of uc.338 and PCBP2 gene. The exons of PCBP2 are indicated by dark gray boxes, and uc.338 is depicted as the light gray box. Primers used for the 5′ RACE were as follows: the antisense intronic (ASI) primer recognized the antisense transcript, and the sense intronic (SI) and nested SI (nSI) primers recognized the sense transcript. The sense exonic (SE) primer recognized PCBP2. Primers used for the 3′ RACE were as follows: the ASI and nASI primers were used to characterize the 3′ UTR of the TUC338 sense transcript, and the antisense exonic (ASE) primer was used to characterize the 3′ UTR of PCBP2. 
           [0024]      FIG. 8 . 5′RACE. HepG2 RNA was retrotranscribed by using the SMARTer RACE cDNA Amplification Kit (Clonetech). The 5′ RACE-ready cDNA was then amplified by PCR using the Universal Primer Mix (UPM) that recognized the SMARTer oligonucleotide added at the 5′ end and the specific gene primers. The SE primer was used as a control to sequence the 5′ end of PCBP2 (band 1). Given the multiple bands obtaind with the SI primer, the PCR product was further amplified witht the nested UPM (nUPM) and nSI primers producing a defined band (band 2). The sequence obtained from the DNA extracted from band 2 is shown. Underline, uc.338 sequence identified by Bejerano et al. (1); bold, exonic sequence; italic, nSI sequences; DNA marker, 1-kb DNA ladder (Promega, Madison, Wis.). 
           [0025]      FIG. 9 . 3′ RACE. HepG2 RNA was polyadenylated and retrotranscribed by using the SMARTer RACE cDNA Amplification Kit. The 3′ RACE-ready cDNA was then amplified by PCR using the UPM and specific gene primers. The ASE primer was used as a control to sequence the 3′ end of PCBP2 (band 3). 3′ RACE-ready cDNA was amplified by a first PCR using the ASI primer, and a second nested PCR with the nASI primer (band 4). The sequence obtained from the DNA extracted from band 4 is shown. Underline shows uc.338(1); capital character, exonic sequence; italic, nSI sequences, DNA marker, 1-kb and 100-bp DNA ladder (Promega, Madison, Wis.). 
           [0026]      FIG. 10 . Cloning of the transcript including uc.338. Panel A: Schematic representation of the transcript including uc.338 (TUC338) in relation to PCBP2 gene. By performing 5′ and 3′ rapid amplificiation of cDNA ends (RACE), we identified 237 nt upstream and 130 nt downstream of the ultraconserved region. The complete sequence of the TUC338 transcript is reported. Panel B: Northern blotting analysis for TUC338 and PCBP2 was performed. TUC338 was expected to be 590 nt long, and PCBP2 was around 1,200 nt long. 
           [0027]      FIG. 11 . TUC338 modulates cell growth in HCC cells. Panel A: HepG2 and Huh-7 cells were transfected with siRNAs against TUC338 or control siRNA for 48 hours and then plated (200,000 viable cells/well) in 6-well plates. After 24, 48 and 72 hours cells were counted by trypan blue staining. Mean values of three independent experiments with SEM are represented. * p&lt;0.05 compared to siRNA control. Cell viability following transfection with siRNA-1 was not significantly different from that with siRNA-2 (p&gt;0.05 at each time point in each of the cell lines). Panel B: HepG2 cells were transfected with siRNA-2 anti-TUC338 or control siRNA for 48 hours, and analysis of cell cycle distribution was performed by flow cytometry. Compared to controls there was a reduction of 30% in S phase and of 12% in G2/M phase for cells transfected with siRNA against TUC338. Bars represent the mean and SEM of three experiments. *p&lt;0.05 compared to controls. Panel C: HepG2 cells were transfected with siRNAs anti-TUC338 or control siRNA by nuclear transfection for 48 hours and then plated in agar in 96-well plates and anchorage-independent growth assessed fluorometrically after 7 days. Bars represent the mean and SEM of 2 experiments performed in seven replicates. *p&lt;0.05. 
           [0028]      FIG. 12 . TUC338 modulates cell growth in mouse hepatocytes. BNL-CL.2 are nonmalignant embryonic mouse hepatocytes. BNL-SV A.8 cells are derived from BNL-CL.2 following SV40 transformation, and exhibit transformed cell growth. Panel A: TUC338 expression was assessed by real-time-PCR and normalized to that of RNU6. Bars represent the mean and standard error of 3 experiments. *p&lt;0.05. Panel B: BNL-CL.2 and BNL-SVA.8 were plated in soft agar and colonies counted after 4 weeks. Representative pictures are shown along with the mean and SEM of three independent experiments. *p&lt;0.05. Panel C: BNL-CL.2 and BNL-SV A.8 cells were plated in 6-well plates and cell number counted by trypan blue staining at different time points. Mean and SEM derived from three independent experiments are shown. *p&lt;0.05 compared to BNL-CL.2. Panel D: BNL-SV A.8 cells were transfected with siRNA-1 anti-TUC338 or siRNA control for 48 hours. Bars represent mean and SEM of four replicates. At the indicated times, cells were counted after trypan blue staining. Mean and SEM from three independent experiments are represented. *p&lt;0.05 compared to siRNA control. 
           [0029]      FIG. 13 . TUC338 expression modulates expression of cell-cycle regulatory proteins. HepG2 and Huh7 cells were serum-starved for 48 hours before transfection with either control siRNA or anti-TUC338 siRNA. Cells were collected 48 hours after transfection, and protein lysates were obtained. Lysates were also obtained from untransfected cells and normal human hepatocytes (HH). Western blotting was performed for the indicated cell-cycle-associated proteins, and their expression was quantitated by densitometry and normalized to that of vinculin. The expression relative to cells transfected with a control siRNA is reported along with representative immunoblots. PCNA is the acronym for proliferating cell nuclear antigen. 
       
    
    
     DESCRIPTION 
       [0030]    Embodiments are based, in part, on the discovery of a functional role for long non-coding RNA genes in cell growth modulation. Embodiments exploit the effects of ucRNAs on tumor cell growth, and demonstrate these RNA genes are useful for studying, diagnosing, and treating cancers, particularly liver cancers. 
         [0031]    The RNA uc.338 is exonic, and represents an alternatively spliced exon of PCBP2, an RNA binding protein involved in mRNA processing. Interestingly, exonic ultraconserved regions are frequently associated with RNA processing such as RNA binding or RNA splicing, suggesting a potential role as regulators of RNA. Despite the exonic location of uc.338 within PCBP2, the expression of these two genes is independent. Advantageously, the examples below demonstrate an important role for uc.338 in human cancer. Various embodiments exploit this novel ucRNA gene as a diagnostic marker and a therapeutic target for HCC. 
         [0032]    Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which these embodiments pertain. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of various embodiments, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. 
         [0033]    The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way. It will be appreciated that there is an implied “about” prior to metrics such as temperatures, concentrations, and times discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings herein. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention. The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. 
         [0034]    As used herein, “cancer” refers to all types of cancers, or neoplasms or benign or malignant tumors. 
         [0035]    The phrase “detecting a cancer” or “diagnosing a cancer” refers to determining the presence or absence of cancer or a precancerous condition in an animal. “Detecting a cancer” also can refer to obtaining indirect evidence regarding the likelihood of the presence of precancerous or cancerous cells in the animal or assessing the predisposition of a patient to the development of a cancer. Detecting a cancer can be accomplished using the methods of this invention alone, in combination with other methods, or in light of other information regarding the state of health of the animal. 
         [0036]    The term “mammal” for purposes of treatment or diagnosis refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. In exemplary embodiments, a mammal is a human. 
         [0037]    A “tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. 
         [0038]    Nucleic acid: a nucleic acid may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively. The present invention contemplates any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition, and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. 
         [0039]    The detection probe may be comprised of naturally occurring or synthetic RNA, DNA, or other oligonucleotides such as an analog of a naturally occurring nucleic acid. At least one locked nucleic acid (LNA) molecule may also be included in the detection probe. A LNA can include a modified RNA nucleotide, for example, in which the ribose moiety of the LNA is modified with an extra bridge connecting the 2′ and 4′ carbons. The bridge may lock the ribose in a 3′-endo structural conformation, thereby enhancing base stability and backbone pre-organization. The LNA may be labeled with a fluorophore. 
         [0040]    As used herein, the term “subject” is intended to include humans and non-human animals. The term “non-human animals” includes all.vertebrates, e.g., mammals and non-mammals, such as non-human primates, pigs, chickens and other birds, mice, dogs, cats, cows, and horses. 
         [0041]    Sequencing: The term sequencing refers to determining the order of nucleotides (base sequences) in a nucleic acid sample, e.g. DNA or RNA. 
         [0042]    Primers: in general, the term primers refer to DNA strands which can prime the synthesis of DNA. DNA polymerase cannot synthesize DNA de novo without primers: it can only extend an existing DNA strand in a reaction in which the complementary strand is used as a template to direct the order of nucleotides to be assembled. Herein, the synthetic oligonucleotide molecules which are used in a polymerase chain reaction (PCR) are referred to as primers. An “SNP-specific primer” is a primer appropriate for oligonucleotide extension at an SNP marker. 
         [0043]    DNA amplification: the term DNA amplification will be typically used to denote the in vitro synthesis of double-stranded DNA molecules using PCR. It is noted that other amplification methods exist and they may be used without departing from the gist. 
         [0044]    In various embodiments, DNA is to be provided. This can be done by methods known in the art per se. The isolation of DNA is generally achieved using common methods in the art such as the collection of tissue from a member of the population, DNA extraction (for instance using the Q-Biogene fast DNA kit), quantification and normalisation to obtain equal amounts of DNA per sample. The DNA can be from a variety of sources (Genomic, RNA, cDNA, BAc, YAC etc.) and organisms (human, mammal, plant, microorganisms, etc.). The isolated DNA may be pooled. 
         [0045]    Also provided are various kits for performing the methods provided herein. Additionally, the kit may include instructional materials for performing various methods presented herein. These instructions may be printed and/or may be supplied, without limitation, as an electronic-readable medium, such as a floppy disc, a CD-ROM, a DVD, a Zip disc, a video cassette, an audiotape, and a flash memory device. Alternatively, instructions may be published on an internet web site or may be distributed to the user as an electronic mail. When a kit is supplied, the different components can be packaged in separate containers. Such packaging of the components separately can permit long term storage without losing the active components&#39; functions. 
         [0046]    In another aspect, the embodiments feature methods of treating a subject who has a disease characterized by abnormal expression of uc.338, as described herein. The methods include administering an inhibitor of uc.338 or TUC338 activity to the subject. In some embodiments, the inhibitor of uc.338 or TUC338 activity can include, for example, one or more of an antisense nucleic acid, a small interfering nucleic acid, etc. The embodiments additionally feature methods of treating a subject having a disease characterized by aberrant cellular proliferation or differentiation, e.g., as described herein. The methods include administering one or more inhibitors of uc.338 activity. In some embodiments, the inhibitor of uc.338 or TUC338 activity includes a nucleic acid molecule described herein. 
         [0047]    The embodiments also provide kits including one or more compounds that selectively bind to an ultraconserved nucleic acid molecule (e.g. uc.338) as described herein, and instructions for use. 
         [0048]    Embodiments include methods for detecting the presence of an uc.338 or TUC338 nucleic acid molecule in a sample. The method includes contacting the sample with a nucleic acid probe or primer that selectively hybridizes to the nucleic acid molecule, and determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample. In some embodiments, the sample comprises RNA molecules and is contacted with a nucleic acid probe. The invention also includes a kit comprising a compound that selectively hybridizes to an ultraconserved nucleic acid molecule (e.g. uc.338), and instructions for use. 
         [0049]    Various embodiments also provide methods for identifying compounds that modulate the expression or activity of a nucleic acid described herein. The method includes contacting the nucleic acid with a test compound; and determining an effect of the test compound on the expression or activity nucleic acid, to thereby identify a compound that modulates the expression or activity of the nucleic acid. 
         [0050]    In another aspect, the invention includes transgenic animals, e.g., animals at least some of whose somatic and germ cells comprise at least one uc.338 transgene. 
         [0051]    Also within the embodiments is the use of uc.338, TUC338, and/or any of the inhibitors of uc.338 or TUC338 activity described herein, e.g., an antisense nucleic acid, a small interfering nucleic acid, or ribozyme, in the manufacture of a medicament for the treatment or prevention of disorders associated with aberrant cellular proliferation. The medicament can be used in a method for treating or preventing disorders associated with aberrant cellular proliferation (e.g., cancer) in a patient suffering from or at risk for a disorder associated with aberrant cellular proliferation. 
         [0052]    Further, within the embodiments is the use of uc.338, TUC338, and/or any of the enhancers of uc.338 or TUC338 activity described herein, e.g., uc.338 nucleic acids, or active fragments thereof, in the manufacture of a medicament for the treatment or prevention of disorders associated with aberrant cellular differentiation and/or proliferation. The medicament can be used in a method for treating or preventing disorders associated with aberrant cellular differentiation and/or proliferation in a patient suffering from or at risk for a disorder associated with aberrant cellular differentiation and/or proliferation. 
         [0053]    Also within the invention are uc.338 and TUC338 nucleic acids, antisense nucleic acids, and small interfering nucleic acids for use in treating disorders associated with aberrant cellular differentiation and/or proliferation. 
       Pharmaceutical Formulations 
       [0054]    The compositions of this invention can be formulated and administered to inhibit a variety of disease states by any means that produces contact of the active ingredient with the agent&#39;s site of action in the body of a mammal. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice. 
         [0055]    Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. The therapeutic compositions of the invention can be formulated for a variety of routes of administration, including systemic and topical or localized administration. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the therapeutic compositions of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank&#39;s solution or Ringer&#39;s solution. In addition, the therapeutic compositions may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. 
         [0056]    For oral administration, the therapeutic compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. 
         [0057]    Preparations for oral administration may be suitably formulated to give controlled release of the active agent. For buccal administration the therapeutic compositions may take the form of tablets or lozenges formulated in a conventional manner. For administration by inhalation, the compositions for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflate or may be formulated containing a powder mix of the therapeutic agents and a suitable powder base such as lactose or starch. 
         [0058]    The therapeutic compositions may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. 
         [0059]    In addition to the formulations described previously, the therapeutic compositions may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the therapeutic compositions may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. 
         [0060]    Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For topical administration, the compositions of the invention are formulated into ointments, salves, gels, or creams as generally known in the art. A wash solution can be used locally to treat an injury or inflammation to accelerate healing. For oral administration, the therapeutic compositions are formulated into conventional oral administration forms such as capsules, tablets, and tonics. 
         [0061]    The therapeutic compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. 
         [0062]    A composition of the present invention can also be formulated as a sustained and/or timed release formulation. Such sustained and/or timed release formulations may be made by sustained release means or delivery devices that are well known to those of ordinary skill in the art, such as those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 4,710,384; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,566, the disclosures of which are each incorporated herein by reference. The pharmaceutical compositions of the present invention can be used to provide slow or sustained release of one or more of the active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like, or a combination thereof to provide the desired release profile in varying proportions. Suitable sustained release formulations known to those of ordinary skill in the art, including those described herein, may be readily selected for use with the pharmaceutical compositions of the invention. Thus, single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gelcaps, caplets, powders, and the like, that are adapted for sustained release are encompassed by the present invention. 
         [0063]    The dosage administered will be a therapeutically effective amount of the compound sufficient to result in amelioration of symptoms of the bone disease and will, of course, vary depending upon known factors such as the pharmacodynamic characteristics of the particular active ingredient and its mode and route of administration; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired. 
         [0064]    Toxicity and therapeutic efficacy of therapeutic compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining The LD50 (The Dose Lethal To 50% Of The Population) and The ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Therapeutic agents which exhibit large therapeutic induces are preferred. While therapeutic compositions that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such therapeutic agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. 
         [0065]    The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any agents used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test therapeutic agent which achieves a half-maximal inhibition of symptoms or inhibition of biochemical activity) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. 
         [0066]    It is understood that appropriate doses of small molecule agents depends upon a number of factors known to those or ordinary skill in the art, e.g., a physician. The dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention. Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. 
         [0067]    These methods described herein are by no means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled artisan. Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect. 
         [0068]    The practice of aspects of the present invention may employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. 
       EXAMPLES 
       [0069]    The following examples are included to demonstrate embodiments. It should be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention. 
       A. General Methods 
       [0070]    Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used, for example, for nucleic acid purification and preparation, chemical analysis, recombinant nucleic acid, and oligonucleotide synthesis. Enzymatic reactions and purification techniques are performed according to manufacturer&#39;s specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the instant specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2000). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of described herein are those well known and commonly used in the art. 
         [0071]    Cell lines. The human HCC cell lines HepG2, PLC/PRF-5, Huh-7, SNU-182, SNU-449, and SK-Hep-1 were obtained from the American Type Culture Collection (Manassas, Va.). Normal human hepatocytes were obtained from Sciencell (San Diego, Calif.). Mouse hepatocyte cell lines BNL-CL.2 and BNL-SVA.8 (SV40-transformed BNL-CL.2 cells) were obtained from the American Type Culture Collection. 
         [0072]    ucRNA expression profiling. RNA was extracted from three separate biological samples for each analysis using Trizol reagent (Invitrogen, Carlsbad, Calif.). Total RNA (5 μg) was reverse transcribed using biotin end-labeled random oligonucleotide primers and cDNA was hybridized to a custom microarray (OSU-CCC 4.0), which includes sense and antisense probes to all 481 human ultraconserved regions (UCRs), each spotted in duplicate. The chromosomal coordinate ranges for all 481 UCRs are listed below in table 3: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 Name 
                 Length 
                 Build 34 (hg16) coords 
               
               
                   
                   
               
             
             
               
                   
                 uc.1 
                 207 
                 chr1: 10307243-10307449 
               
               
                   
                 uc.2 
                 207 
                 chr1: 10442089-10442295 
               
               
                   
                 uc.3 
                 225 
                 chr1: 10460711-10460935 
               
               
                   
                 uc.4 
                 359 
                 chr1: 10467795-10468153 
               
               
                   
                 uc.5 
                 214 
                 chr1: 10490897-10491110 
               
               
                   
                 uc.6 
                 301 
                 chr1: 10504667-10504967 
               
               
                   
                 uc.7 
                 256 
                 chr1: 10545679-10545934 
               
               
                   
                 uc.8 
                 216 
                 chr1: 10561364-10561579 
               
               
                   
                 uc.9 
                 202 
                 chr1: 10634956-10635157 
               
               
                   
                 uc.10 
                 275 
                 chr1: 10675120-10675394 
               
               
                   
                 uc.12 
                 201 
                 chr1: 35077889-35078089 
               
               
                   
                 uc.13 
                 237 
                 chr1: 35786852-35787088 
               
               
                   
                 uc.14 
                 213 
                 chr1: 37908298-37908510 
               
               
                   
                 uc.15 
                 233 
                 chr1: 37974370-37974602 
               
               
                   
                 uc.16 
                 211 
                 chr1: 38041493-38041703 
               
               
                   
                 uc.17 
                 237 
                 chr1: 38215445-38215681 
               
               
                   
                 uc.18 
                 238 
                 chr1: 44128955-44129192 
               
               
                   
                 uc.19 
                 256 
                 chr1: 44403606-44403861 
               
               
                   
                 uc.20 
                 269 
                 chr1: 44415666-44415934 
               
               
                   
                 uc.21 
                 235 
                 chr1: 48482903-48483137 
               
               
                   
                 uc.22 
                 308 
                 chr1: 50376149-50376456 
               
               
                   
                 uc.23 
                 229 
                 chr1: 50405695-50405923 
               
               
                   
                 uc.24 
                 336 
                 chr1: 50469063-50469398 
               
               
                   
                 uc.25 
                 235 
                 chr1: 50535952-50536186 
               
               
                   
                 uc.26 
                 212 
                 chr1: 62739563-62739774 
               
               
                   
                 uc.27 
                 290 
                 chr1: 62739797-62740086 
               
               
                   
                 uc.28 
                 355 
                 chr1: 70066629-70066983 
               
               
                   
                 uc.29 
                 219 
                 chr1: 87244976-87245194 
               
               
                   
                 uc.30 
                 243 
                 chr1: 87451593-87451835 
               
               
                   
                 uc.31 
                 253 
                 chr1: 88395168-88395420 
               
               
                   
                 uc.33 
                 312 
                 chr1: 96743538-96743849 
               
               
                   
                 uc.34 
                 208 
                 chr1: 96751973-96752180 
               
               
                   
                 uc.35 
                 205 
                 chr1: 97465205-97465409 
               
               
                   
                 uc.36 
                 264 
                 chr1: 108587040-108587303 
               
               
                   
                 uc.37 
                 202 
                 chr1: 114578780-114578981 
               
               
                   
                 uc.38 
                 224 
                 chr1: 161127332-161127555 
               
               
                   
                 uc.39 
                 356 
                 chr1: 161210912-161211267 
               
               
                   
                 uc.40 
                 247 
                 chr1: 161825339-161825585 
               
               
                   
                 uc.41 
                 216 
                 chr1: 210655265-210655480 
               
               
                   
                 uc.42 
                 266 
                 chr1: 212945530-212945795 
               
               
                   
                 uc.43 
                 257 
                 chr1: 240842759-240843015 
               
               
                   
                 uc.44 
                 230 
                 chr1: 241164431-241164660 
               
               
                   
                 uc.45 
                 203 
                 chr1: 241963410-241963612 
               
               
                   
                 uc.46 
                 217 
                 chr1: 241964327-241964543 
               
               
                   
                 uc.47 
                 227 
                 chr2: 7796390-7796616 
               
               
                   
                 uc.48 
                 298 
                 chr2: 20462845-20463142 
               
               
                   
                 uc.49 
                 207 
                 chr2: 33787944-33788150 
               
               
                   
                 uc.50 
                 222 
                 chr2: 38950836-38951057 
               
               
                   
                 uc.51 
                 207 
                 chr2: 57947093-57947299 
               
               
                   
                 uc.52 
                 274 
                 chr2: 59082720-59082993 
               
               
                   
                 uc.53 
                 232 
                 chr2: 59107845-59108076 
               
               
                   
                 uc.54 
                 209 
                 chr2: 59173937-59174145 
               
               
                   
                 uc.55 
                 240 
                 chr2: 59721112-59721351 
               
               
                   
                 uc.56 
                 202 
                 chr2: 59922365-59922566 
               
               
                   
                 uc.57 
                 246 
                 chr2: 60113774-60114019 
               
               
                   
                 uc.58 
                 203 
                 chr2: 60272497-60272699 
               
               
                   
                 uc.59 
                 220 
                 chr2: 60272917-60273136 
               
               
                   
                 uc.60 
                 217 
                 chr2: 60416094-60416310 
               
               
                   
                 uc.61 
                 326 
                 chr2: 60662107-60662432 
               
               
                   
                 uc.62 
                 234 
                 chr2: 60755216-60755449 
               
               
                   
                 uc.63 
                 278 
                 chr2: 61727035-61727312 
               
               
                   
                 uc.64 
                 245 
                 chr2: 63168625-63168869 
               
               
                   
                 uc.65 
                 212 
                 chr2: 66273125-66273336 
               
               
                   
                 uc.66 
                 247 
                 chr2: 73149541-73149787 
               
               
                   
                 uc.67 
                 217 
                 chr2: 104358126-104358342 
               
               
                   
                 uc.68 
                 255 
                 chr2: 144125491-144125745 
               
               
                   
                 uc.69 
                 301 
                 chr2: 144322879-144323179 
               
               
                   
                 uc.70 
                 237 
                 chr2: 144648108-144648344 
               
               
                   
                 uc.71 
                 248 
                 chr2: 144923625-144923872 
               
               
                   
                 uc.72 
                 407 
                 chr2: 144925741-144926147 
               
               
                   
                 uc.73 
                 201 
                 chr2: 144973082-144973282 
               
               
                   
                 uc.74 
                 538 
                 chr2: 145036732-145037269 
               
               
                   
                 uc.75 
                 236 
                 chr2: 145356619-145356854 
               
               
                   
                 uc.76 
                 335 
                 chr2: 145371902-145372236 
               
               
                   
                 uc.77 
                 296 
                 chr2: 145396534-145396829 
               
               
                   
                 uc.78 
                 248 
                 chr2: 145399123-145399370 
               
               
                   
                 uc.79 
                 295 
                 chr2: 145407946-145408240 
               
               
                   
                 uc.80 
                 294 
                 chr2: 145411621-145411914 
               
               
                   
                 uc.81 
                 211 
                 chr2: 147344834-147345044 
               
               
                   
                 uc.82 
                 210 
                 chr2: 156929644-156929853 
               
               
                   
                 uc.83 
                 296 
                 chr2: 157194172-157194467 
               
               
                   
                 uc.84 
                 209 
                 chr2: 157397251-157397459 
               
               
                   
                 uc.85 
                 248 
                 chr2: 157753959-157754206 
               
               
                   
                 uc.86 
                 340 
                 chr2: 157862655-157862994 
               
               
                   
                 uc.87 
                 290 
                 chr2: 158102814-158103103 
               
               
                   
                 uc.88 
                 312 
                 chr2: 162297586-162297897 
               
               
                   
                 uc.89 
                 307 
                 chr2: 162441217-162441523 
               
               
                   
                 uc.90 
                 206 
                 chr2: 162475571-162475776 
               
               
                   
                 uc.91 
                 207 
                 chr2: 163247566-163247772 
               
               
                   
                 uc.92 
                 309 
                 chr2: 164653223-164653531 
               
               
                   
                 uc.93 
                 263 
                 chr2: 164864451-164864713 
               
               
                   
                 uc.94 
                 200 
                 chr2: 165046714-165046913 
               
               
                   
                 uc.95 
                 251 
                 chr2: 171774074-171774324 
               
               
                   
                 uc.96 
                 261 
                 chr2: 173023218-173023478 
               
               
                   
                 uc.97 
                 442 
                 chr2: 173025175-173025616 
               
               
                   
                 uc.98 
                 238 
                 chr2: 173159062-173159299 
               
               
                   
                 uc.99 
                 398 
                 chr2: 173160925-173161322 
               
               
                   
                 uc.100 
                 207 
                 chr2: 174317324-174317530 
               
               
                   
                 uc.101 
                 254 
                 chr2: 174977025-174977278 
               
               
                   
                 uc.102 
                 338 
                 chr2: 175148953-175149290 
               
               
                   
                 uc.103 
                 233 
                 chr2: 175172216-175172448 
               
               
                   
                 uc.104 
                 216 
                 chr2: 175189478-175189693 
               
               
                   
                 uc.105 
                 223 
                 chr2: 175192331-175192553 
               
               
                   
                 uc.106 
                 206 
                 chr2: 175227967-175228172 
               
               
                   
                 uc.107 
                 237 
                 chr2: 175410152-175410388 
               
               
                   
                 uc.108 
                 374 
                 chr2: 177142901-177143274 
               
               
                   
                 uc.109 
                 224 
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                 uc.110 
                 243 
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                 uc.111 
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                 uc.483 
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                 uc.112 
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                 uc.113 
                 247 
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                 uc.114 
                 294 
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                 uc.115 
                 219 
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                 uc.116 
                 206 
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                 uc.117 
                 251 
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                 uc.118 
                 219 
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                 uc.119 
                 301 
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                 uc.120 
                 270 
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                 uc.121 
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                 uc.122 
                 215 
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                 uc.123 
                 492 
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                 uc.124 
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                 uc.125 
                 265 
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                 uc.126 
                 271 
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                 uc.127 
                 272 
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                 uc.128 
                 299 
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                 uc.129 
                 212 
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                 uc.130 
                 224 
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                 uc.131 
                 207 
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                 uc.132 
                 208 
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                 uc.133 
                 277 
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                 uc.134 
                 211 
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                 uc.135 
                 201 
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                 uc.136 
                 347 
                 chr3: 170514865-170515211 
               
               
                   
                 uc.137 
                 385 
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                 uc.138 
                 419 
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                 uc.139 
                 338 
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                 uc.140 
                 223 
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                 uc.141 
                 295 
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                 uc.142 
                 259 
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                 uc.143 
                 218 
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                 uc.144 
                 205 
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                 uc.145 
                 248 
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                 uc.146 
                 214 
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                 uc.147 
                 308 
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                 uc.148 
                 240 
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                 uc.149 
                 204 
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                 uc.150 
                 262 
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                 uc.151 
                 214 
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                 uc.152 
                 201 
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                 uc.153 
                 240 
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                 uc.154 
                 203 
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                 uc.155 
                 207 
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                 uc.156 
                 213 
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                 uc.157 
                 207 
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                 uc.158 
                 224 
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                 uc.159 
                 472 
                 chr5: 77232005-77232476 
               
               
                   
                 uc.160 
                 322 
                 chr5: 77352917-77353238 
               
               
                   
                 uc.161 
                 278 
                 chr5: 77442602-77442879 
               
               
                   
                 uc.162 
                 218 
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                 uc.163 
                 376 
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                 uc.164 
                 203 
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                 uc.165 
                 224 
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                 uc.166 
                 310 
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                 uc.167 
                 201 
                 chr5: 88263697-88263897 
               
               
                   
                 uc.168 
                 220 
                 chr5: 91012866-91013085 
               
               
                   
                 uc.169 
                 204 
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                 uc.170 
                 310 
                 chr5: 93301743-93302052 
               
               
                   
                 uc.171 
                 208 
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                 uc.172 
                 218 
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                 uc.173 
                 276 
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                 uc.174 
                 260 
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                 uc.175 
                 250 
                 chr5: 158322733-158322982 
               
               
                   
                 uc.176 
                 246 
                 chr5: 167313590-167313835 
               
               
                   
                 uc.177 
                 257 
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                 uc.178 
                 249 
                 chr5: 170398920-170399168 
               
               
                   
                 uc.179 
                 219 
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                 uc.180 
                 225 
                 chr5: 170609411-170609635 
               
               
                   
                 uc.181 
                 278 
                 chr5: 170610401-170610678 
               
               
                   
                 uc.182 
                 239 
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                 uc.183 
                 236 
                 chr5: 171365442-171365677 
               
               
                   
                 uc.184 
                 230 
                 chr5: 173366215-173366444 
               
               
                   
                 uc.185 
                 411 
                 chr5: 178157908-178158318 
               
               
                   
                 uc.186 
                 305 
                 chr5: 179155889-179156193 
               
               
                   
                 uc.187 
                 212 
                 chr6: 10502663-10502874 
               
               
                   
                 uc.188 
                 215 
                 chr6: 16407363-16407577 
               
               
                   
                 uc.189 
                 573 
                 chr6: 36614372-36614944 
               
               
                   
                 uc.190 
                 200 
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                 uc.191 
                 208 
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                 uc.192 
                 244 
                 chr6: 51195833-51196076 
               
               
                   
                 uc.193 
                 319 
                 chr6: 86317282-86317600 
               
               
                   
                 uc.194 
                 201 
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                 uc.195 
                 273 
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                 uc.196 
                 221 
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                 uc.197 
                 224 
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                 uc.198 
                 307 
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                 uc.199 
                 256 
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                 uc.200 
                 254 
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                 uc.201 
                 240 
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                 uc.202 
                 230 
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                 uc.203 
                 203 
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                 uc.204 
                 202 
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                 uc.205 
                 252 
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                 uc.206 
                 499 
                 chr7: 20748081-20748579 
               
               
                   
                 uc.207 
                 230 
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                 uc.208 
                 218 
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                 uc.209 
                 250 
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                 uc.210 
                 257 
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                 uc.211 
                 291 
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                 uc.212 
                 205 
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                 uc.213 
                 201 
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                 uc.214 
                 243 
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                 uc.215 
                 262 
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                 uc.216 
                 312 
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                 uc.217 
                 221 
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                 uc.218 
                 286 
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                 uc.219 
                 210 
                 chr7: 69392897-69393106 
               
               
                   
                 uc.220 
                 257 
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                 uc.221 
                 349 
                 chr7: 96253032-96253380 
               
               
                   
                 uc.222 
                 201 
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                 uc.223 
                 268 
                 chr7: 113612688-113612955 
               
               
                   
                 uc.224 
                 295 
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                 uc.225 
                 201 
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                 uc.226 
                 205 
                 chr7: 113763821-113764025 
               
               
                   
                 uc.227 
                 231 
                 chr7: 113849819-113850049 
               
               
                   
                 uc.228 
                 265 
                 chr7: 114671200-114671464 
               
               
                   
                 uc.229 
                 296 
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                 uc.230 
                 238 
                 chr7: 114873964-114874201 
               
               
                   
                 uc.231 
                 224 
                 chr7: 115136620-115136843 
               
               
                   
                 uc.232 
                 247 
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                 uc.233 
                 266 
                 chr7: 150220055-150220320 
               
               
                   
                 uc.234 
                 272 
                 chr7: 156229240-156229511 
               
               
                   
                 uc.235 
                 227 
                 chr8: 25797831-25798057 
               
               
                   
                 uc.236 
                 267 
                 chr8: 37307753-37308019 
               
               
                   
                 uc.237 
                 468 
                 chr8: 53187862-53188329 
               
               
                   
                 uc.238 
                 358 
                 chr8: 53217077-53217434 
               
               
                   
                 uc.239 
                 300 
                 chr8: 59992334-59992633 
               
               
                   
                 uc.240 
                 206 
                 chr8: 65542500-65542705 
               
               
                   
                 uc.241 
                 202 
                 chr8: 65547091-65547292 
               
               
                   
                 uc.242 
                 265 
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                 uc.243 
                 216 
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                 uc.244 
                 321 
                 chr8: 105918932-105919252 
               
               
                   
                 uc.245 
                 339 
                 chr8: 106290415-106290753 
               
               
                   
                 uc.246 
                 284 
                 chr8: 119079806-119080089 
               
               
                   
                 uc.247 
                 361 
                 chr9: 959154-959514 
               
               
                   
                 uc.248 
                 222 
                 chr9: 964189-964410 
               
               
                   
                 uc.249 
                 260 
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                 uc.250 
                 209 
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                 uc.251 
                 213 
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                 uc.252 
                 231 
                 chr9: 16700753-16700983 
               
               
                   
                 uc.253 
                 222 
                 chr9: 17322212-17322433 
               
               
                   
                 uc.254 
                 279 
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                 uc.255 
                 232 
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                 uc.256 
                 206 
                 chr9: 23682234-23682439 
               
               
                   
                 uc.257 
                 264 
                 chr9: 37205204-37205467 
               
               
                   
                 uc.258 
                 201 
                 chr9: 37314424-37314624 
               
               
                   
                 uc.259 
                 308 
                 chr9: 75084998-75085305 
               
               
                   
                 uc.260 
                 231 
                 chr9: 76929543-76929773 
               
               
                   
                 uc.261 
                 311 
                 chr9: 77328693-77329003 
               
               
                   
                 uc.262 
                 255 
                 chr9: 79184841-79185095 
               
               
                   
                 uc.263 
                 207 
                 chr9: 82047403-82047609 
               
               
                   
                 uc.264 
                 267 
                 chr9: 82047611-82047877 
               
               
                   
                 uc.265 
                 217 
                 chr9: 103498309-103498525 
               
               
                   
                 uc.266 
                 243 
                 chr9: 104758130-104758372 
               
               
                   
                 uc.267 
                 203 
                 chr9: 120429935-120430137 
               
               
                   
                 uc.268 
                 251 
                 chr9: 120982873-120983123 
               
               
                   
                 uc.269 
                 217 
                 chr9: 121913982-121914198 
               
               
                   
                 uc.270 
                 278 
                 chr9: 123680114-123680391 
               
               
                   
                 uc.271 
                 211 
                 chr9: 123680397-123680607 
               
               
                   
                 uc.272 
                 213 
                 chr9: 123808633-123808845 
               
               
                   
                 uc.273 
                 321 
                 chr9: 123893643-123893963 
               
               
                   
                 uc.274 
                 327 
                 chr9: 123897916-123898242 
               
               
                   
                 uc.275 
                 255 
                 chr9: 123960161-123960415 
               
               
                   
                 uc.276 
                 432 
                 chr9: 123981857-123982288 
               
               
                   
                 uc.277 
                 276 
                 chr9: 123983755-123984030 
               
               
                   
                 uc.278 
                 237 
                 chr9: 124022210-124022446 
               
               
                   
                 uc.279 
                 336 
                 chr9: 124048604-124048939 
               
               
                   
                 uc.280 
                 220 
                 chr9: 124054051-124054270 
               
               
                   
                 uc.281 
                 238 
                 chr9: 130771570-130771807 
               
               
                   
                 uc.282 
                 207 
                 chr9: 135399783-135399989 
               
               
                   
                 uc.283 
                 277 
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                 uc.284 
                 209 
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                 uc.285 
                 232 
                 chr10: 69860594-69860825 
               
               
                   
                 uc.286 
                 252 
                 chr10: 76483627-76483878 
               
               
                   
                 uc.287 
                 257 
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                 uc.288 
                 223 
                 chr10: 77071786-77072008 
               
               
                   
                 uc.289 
                 254 
                 chr10: 77335142-77335395 
               
               
                   
                 uc.290 
                 206 
                 chr10: 77386885-77387090 
               
               
                   
                 uc.291 
                 231 
                 chr10: 77628227-77628457 
               
               
                   
                 uc.292 
                 217 
                 chr10: 98380042-98380258 
               
               
                   
                 uc.293 
                 243 
                 chr10: 102037256-102037498 
               
               
                   
                 uc.294 
                 444 
                 chr10: 102038204-102038647 
               
               
                   
                 uc.295 
                 209 
                 chr10: 102039687-102039895 
               
               
                   
                 uc.296 
                 461 
                 chr10: 102079693-102080153 
               
               
                   
                 uc.297 
                 364 
                 chr10: 102083807-102084170 
               
               
                   
                 uc.298 
                 359 
                 chr10: 102112245-102112603 
               
               
                   
                 uc.299 
                 210 
                 chr10: 102174022-102174231 
               
               
                   
                 uc.300 
                 208 
                 chr10: 102211705-102211912 
               
               
                   
                 uc.301 
                 284 
                 chr10: 102232378-102232661 
               
               
                   
                 uc.302 
                 341 
                 chr10: 102643767-102644107 
               
               
                   
                 uc.303 
                 272 
                 chr10: 102717014-102717285 
               
               
                   
                 uc.304 
                 272 
                 chr10: 102747091-102747362 
               
               
                   
                 uc.305 
                 305 
                 chr10: 102876022-102876326 
               
               
                   
                 uc.306 
                 224 
                 chr10: 102876626-102876849 
               
               
                   
                 uc.307 
                 232 
                 chr10: 102908570-102908801 
               
               
                   
                 uc.308 
                 277 
                 chr10: 102910399-102910675 
               
               
                   
                 uc.309 
                 268 
                 chr10: 102931618-102931885 
               
               
                   
                 uc.310 
                 229 
                 chr10: 114068810-114069038 
               
               
                   
                 uc.311 
                 219 
                 chr10: 119738989-119739207 
               
               
                   
                 uc.312 
                 322 
                 chr10: 119741124-119741445 
               
               
                   
                 uc.313 
                 231 
                 chr10: 121004761-121004991 
               
               
                   
                 uc.314 
                 202 
                 chr10: 124392535-124392736 
               
               
                   
                 uc.315 
                 235 
                 chr10: 124392947-124393181 
               
               
                   
                 uc.316 
                 240 
                 chr10: 126479965-126480204 
               
               
                   
                 uc.317 
                 218 
                 chr10: 130920738-130920955 
               
               
                   
                 uc.318 
                 321 
                 chr10: 131165986-131166306 
               
               
                   
                 uc.319 
                 316 
                 chr11: 8269004-8269319 
               
               
                   
                 uc.320 
                 335 
                 chr11: 8282143-8282477 
               
               
                   
                 uc.321 
                 204 
                 chr11: 15588733-15588936 
               
               
                   
                 uc.322 
                 223 
                 chr11: 16280660-16280882 
               
               
                   
                 uc.323 
                 200 
                 chr11: 16439645-16439844 
               
               
                   
                 uc.324 
                 225 
                 chr11: 30521830-30522054 
               
               
                   
                 uc.325 
                 235 
                 chr11: 31649953-31650187 
               
               
                   
                 uc.326 
                 315 
                 chr11: 31749989-31750303 
               
               
                   
                 uc.327 
                 268 
                 chr11: 31750593-31750860 
               
               
                   
                 uc.328 
                 231 
                 chr11: 31789972-31790202 
               
               
                   
                 uc.329 
                 307 
                 chr11: 32162301-32162607 
               
               
                   
                 uc.330 
                 207 
                 chr11: 66169256-66169462 
               
               
                   
                 uc.331 
                 218 
                 chr11: 82921467-82921684 
               
               
                   
                 uc.332 
                 337 
                 chr11: 115770342-115770678 
               
               
                   
                 uc.333 
                 270 
                 chr11: 124182299-124182568 
               
               
                   
                 uc.334 
                 222 
                 chr11: 131405597-131405818 
               
               
                   
                 uc.335 
                 214 
                 chr12: 16606681-16606894 
               
               
                   
                 uc.336 
                 251 
                 chr12: 24183273-24183523 
               
               
                   
                 uc.337 
                 218 
                 chr12: 40035446-40035663 
               
               
                   
                 uc.338 
                 223 
                 chr12: 52144756-52144978 
               
               
                   
                 uc.339 
                 252 
                 chr12: 52357363-52357614 
               
               
                   
                 uc.340 
                 259 
                 chr12: 52377099-52377357 
               
               
                   
                 uc.341 
                 314 
                 chr12: 52669185-52669498 
               
               
                   
                 uc.342 
                 227 
                 chr12: 52696761-52696987 
               
               
                   
                 uc.343 
                 388 
                 chr12: 52708708-52709095 
               
               
                   
                 uc.344 
                 254 
                 chr12: 52713153-52713406 
               
               
                   
                 uc.345 
                 301 
                 chr12: 52733867-52734167 
               
               
                   
                 uc.346 
                 202 
                 chr12: 105478977-105479178 
               
               
                   
                 uc.347 
                 209 
                 chr13: 69591989-69592197 
               
               
                   
                 uc.348 
                 240 
                 chr13: 69861358-69861597 
               
               
                   
                 uc.349 
                 203 
                 chr13: 69919303-69919505 
               
               
                   
                 uc.350 
                 240 
                 chr13: 70054101-70054340 
               
               
                   
                 uc.351 
                 255 
                 chr13: 70466901-70467155 
               
               
                   
                 uc.352 
                 200 
                 chr13: 70492166-70492365 
               
               
                   
                 uc.353 
                 323 
                 chr13: 70569554-70569876 
               
               
                   
                 uc.354 
                 235 
                 chr13: 76774830-76775064 
               
               
                   
                 uc.355 
                 228 
                 chr13: 93316883-93317110 
               
               
                   
                 uc.356 
                 251 
                 chr13: 95706821-95707071 
               
               
                   
                 uc.357 
                 242 
                 chr13: 110664338-110664579 
               
               
                   
                 uc.358 
                 226 
                 chr14: 24368162-24368387 
               
               
                   
                 uc.359 
                 324 
                 chr14: 24905096-24905419 
               
               
                   
                 uc.360 
                 287 
                 chr14: 24905510-24905796 
               
               
                   
                 uc.361 
                 267 
                 chr14: 27223174-27223440 
               
               
                   
                 uc.362 
                 239 
                 chr14: 27338791-27339029 
               
               
                   
                 uc.363 
                 265 
                 chr14: 27851358-27851622 
               
               
                   
                 uc.364 
                 207 
                 chr14: 28702798-28703004 
               
               
                   
                 uc.365 
                 278 
                 chr14: 28732401-28732678 
               
               
                   
                 uc.366 
                 202 
                 chr14: 29372746-29372947 
               
               
                   
                 uc.367 
                 298 
                 chr14: 31834502-31834799 
               
               
                   
                 uc.368 
                 228 
                 chr14: 32058615-32058842 
               
               
                   
                 uc.369 
                 213 
                 chr14: 32112656-32112868 
               
               
                   
                 uc.370 
                 393 
                 chr14: 32192610-32193002 
               
               
                   
                 uc.371 
                 296 
                 chr14: 34010228-34010523 
               
               
                   
                 uc.372 
                 277 
                 chr14: 34033074-34033350 
               
               
                   
                 uc.373 
                 394 
                 chr14: 34571846-34572239 
               
               
                   
                 uc.374 
                 224 
                 chr14: 35705952-35706175 
               
               
                   
                 uc.375 
                 300 
                 chr14: 35767259-35767558 
               
               
                   
                 uc.376 
                 290 
                 chr14: 43555788-43556077 
               
               
                   
                 uc.377 
                 217 
                 chr14: 43569061-43569277 
               
               
                   
                 uc.378 
                 251 
                 chr14: 78317518-78317768 
               
               
                   
                 uc.379 
                 252 
                 chr14: 95421409-95421660 
               
               
                   
                 uc.380 
                 232 
                 chr14: 95752635-95752866 
               
               
                   
                 uc.381 
                 238 
                 chr14: 95869331-95869568 
               
               
                   
                 uc.382 
                 200 
                 chr15: 33634968-33635167 
               
               
                   
                 uc.383 
                 269 
                 chr15: 34535983-34536251 
               
               
                   
                 uc.384 
                 266 
                 chr15: 34681449-34681714 
               
               
                   
                 uc.385 
                 209 
                 chr15: 34901726-34901934 
               
               
                   
                 uc.386 
                 203 
                 chr15: 35238065-35238267 
               
               
                   
                 uc.387 
                 238 
                 chr15: 39748110-39748347 
               
               
                   
                 uc.388 
                 298 
                 chr15: 55141581-55141878 
               
               
                   
                 uc.389 
                 271 
                 chr15: 65381203-65381473 
               
               
                   
                 uc.390 
                 205 
                 chr15: 65593988-65594192 
               
               
                   
                 uc.391 
                 311 
                 chr15: 65756122-65756432 
               
               
                   
                 uc.392 
                 256 
                 chr15: 68107952-68108207 
               
               
                   
                 uc.393 
                 275 
                 chr15: 72630059-72630333 
               
               
                   
                 uc.394 
                 202 
                 chr15: 94965868-94966069 
               
               
                   
                 uc.395 
                 249 
                 chr16: 24545554-24545802 
               
               
                   
                 uc.396 
                 208 
                 chr16: 48872692-48872899 
               
               
                   
                 uc.397 
                 311 
                 chr16: 49513876-49514186 
               
               
                   
                 uc.398 
                 322 
                 chr16: 49668919-49669240 
               
               
                   
                 uc.399 
                 214 
                 chr16: 50627024-50627237 
               
               
                   
                 uc.400 
                 206 
                 chr16: 51450088-51450293 
               
               
                   
                 uc.401 
                 250 
                 chr16: 52273300-52273549 
               
               
                   
                 uc.402 
                 245 
                 chr16: 53432987-53433231 
               
               
                   
                 uc.403 
                 206 
                 chr16: 54102457-54102662 
               
               
                   
                 uc.404 
                 235 
                 chr16: 55001959-55002193 
               
               
                   
                 uc.405 
                 210 
                 chr16: 59350792-59351001 
               
               
                   
                 uc.406 
                 211 
                 chr16: 69456552-69456762 
               
               
                   
                 uc.407 
                 326 
                 chr16: 69457343-69457668 
               
               
                   
                 uc.408 
                 252 
                 chr16: 72597321-72597572 
               
               
                   
                 uc.409 
                 244 
                 chr16: 72869147-72869390 
               
               
                   
                 uc.410 
                 219 
                 chr17: 35207780-35207998 
               
               
                   
                 uc.411 
                 229 
                 chr17: 35525169-35525397 
               
               
                   
                 uc.412 
                 268 
                 chr17: 35532036-35532303 
               
               
                   
                 uc.413 
                 272 
                 chr17: 37941482-37941753 
               
               
                   
                 uc.414 
                 246 
                 chr17: 38624137-38624382 
               
               
                   
                 uc.415 
                 207 
                 chr17: 47138544-47138750 
               
               
                   
                 uc.416 
                 286 
                 chr17: 47145525-47145810 
               
               
                   
                 uc.417 
                 222 
                 chr17: 47156950-47157171 
               
               
                   
                 uc.418 
                 217 
                 chr17: 56556866-56557082 
               
               
                   
                 uc.419 
                 289 
                 chr17: 56557353-56557641 
               
               
                   
                 uc.420 
                 233 
                 chr17: 63046872-63047104 
               
               
                   
                 uc.421 
                 345 
                 chr18: 20945142-20945486 
               
               
                   
                 uc.422 
                 226 
                 chr18: 21000175-21000400 
               
               
                   
                 uc.423 
                 223 
                 chr18: 21008342-21008564 
               
               
                   
                 uc.424 
                 215 
                 chr18: 21019766-21019980 
               
               
                   
                 uc.425 
                 325 
                 chr18: 21117194-21117518 
               
               
                   
                 uc.426 
                 262 
                 chr18: 22167579-22167840 
               
               
                   
                 uc.427 
                 215 
                 chr18: 22489186-22489400 
               
               
                   
                 uc.428 
                 239 
                 chr18: 28605196-28605434 
               
               
                   
                 uc.429 
                 238 
                 chr18: 32732528-32732765 
               
               
                   
                 uc.430 
                 213 
                 chr18: 33315637-33315849 
               
               
                   
                 uc.431 
                 230 
                 chr18: 33430587-33430816 
               
               
                   
                 uc.432 
                 211 
                 chr18: 33816919-33817129 
               
               
                   
                 uc.433 
                 206 
                 chr18: 34315619-34315824 
               
               
                   
                 uc.434 
                 249 
                 chr18: 43022775-43023023 
               
               
                   
                 uc.435 
                 227 
                 chr18: 51238918-51239144 
               
               
                   
                 uc.436 
                 210 
                 chr18: 51403228-51403437 
               
               
                   
                 uc.437 
                 215 
                 chr18: 70484635-70484849 
               
               
                   
                 uc.438 
                 241 
                 chr18: 70484851-70485091 
               
               
                   
                 uc.439 
                 263 
                 chr18: 70490008-70490270 
               
               
                   
                 uc.440 
                 329 
                 chr18: 70490342-70490670 
               
               
                   
                 uc.441 
                 248 
                 chr18: 70695569-70695816 
               
               
                   
                 uc.442 
                 249 
                 chr18: 70719689-70719937 
               
               
                   
                 uc.443 
                 239 
                 chr19: 8433269-8433507 
               
               
                   
                 uc.444 
                 388 
                 chr19: 35186619-35187006 
               
               
                   
                 uc.445 
                 310 
                 chr19: 35258275-35258584 
               
               
                   
                 uc.446 
                 272 
                 chr19: 35439694-35439965 
               
               
                   
                 uc.447 
                 273 
                 chr19: 35459621-35459893 
               
               
                   
                 uc.448 
                 232 
                 chr19: 35533370-35533601 
               
               
                   
                 uc.449 
                 290 
                 chr19: 35695381-35695670 
               
               
                   
                 uc.450 
                 211 
                 chr19: 36279466-36279676 
               
               
                   
                 uc.451 
                 225 
                 chr19: 36498460-36498684 
               
               
                   
                 uc.452 
                 204 
                 chr19: 36519787-36519990 
               
               
                   
                 uc.453 
                 325 
                 chr19: 47129157-47129481 
               
               
                   
                 uc.454 
                 208 
                 chr20: 4861440-4861647 
               
               
                   
                 uc.455 
                 245 
                 chr20: 35043808-35044052 
               
               
                   
                 uc.456 
                 320 
                 chr20: 42773185-42773504 
               
               
                   
                 uc.457 
                 211 
                 chr22: 17770463-17770673 
               
               
                   
                 uc.458 
                 204 
                 chr22: 34420305-34420508 
               
               
                   
                 uc.459 
                 255 
                 chrX: 20895986-20896240 
               
               
                   
                 uc.460 
                 275 
                 chrX: 24184937-24185211 
               
               
                   
                 uc.461 
                 397 
                 chrX: 24226223-24226619 
               
               
                   
                 uc.462 
                 779 
                 chrX: 24256252-24257030 
               
               
                   
                 uc.463 
                 275 
                 chrX: 24277308-24277582 
               
               
                   
                 uc.464 
                 770 
                 chrX: 24277584-24278353 
               
               
                   
                 uc.465 
                 310 
                 chrX: 24278907-24279216 
               
               
                   
                 uc.466 
                 349 
                 chrX: 24307884-24308232 
               
               
                   
                 uc.467 
                 731 
                 chrX: 24369780-24370510 
               
               
                   
                 uc.468 
                 489 
                 chrX: 24378989-24379477 
               
               
                   
                 uc.469 
                 222 
                 chrX: 24379479-24379700 
               
               
                   
                 uc.470 
                 341 
                 chrX: 24762642-24762982 
               
               
                   
                 uc.471 
                 239 
                 chrX: 40239309-40239547 
               
               
                   
                 uc.472 
                 202 
                 chrX: 40410244-40410445 
               
               
                   
                 uc.473 
                 222 
                 chrX: 69240015-69240236 
               
               
                   
                 uc.474 
                 210 
                 chrX: 69335634-69335843 
               
               
                   
                 uc.475 
                 397 
                 chrX: 69632846-69633242 
               
               
                   
                 uc.476 
                 238 
                 chrX: 80545473-80545710 
               
               
                   
                 uc.477 
                 209 
                 chrX: 101813348-101813556 
               
               
                   
                 uc.478 
                 252 
                 chrX: 121297212-121297463 
               
               
                   
                 uc.479 
                 302 
                 chrX: 121311506-121311807 
               
               
                   
                 uc.480 
                 202 
                 chrX: 121933027-121933228 
               
               
                   
                 uc.481 
                 204 
                 chrX: 121933230-121933433 
               
               
                   
                   
               
             
          
         
       
     
         [0073]    Sequence information for all 481 UCRs is available at http://genome.ucsc.edu/cgi-bin/hgGateway (July 2003 human reference sequence (NCBI Build 34) was produced by the International Human Genome Sequencing Consortium). 
         [0074]    Biotin-containing transcripts were detected using streptavidin—Alexa647 conjugate, scanned and analyzed using an Axon 4000B scanner and the GenePix 6.0 software (Axon Instruments, Downingtown, Pa.). The mean fluorescence intensity of replicate spots were subtracted from background and normalized using the global median method. We selected ucRNAs measured as present in all the three replicates. Differentially expressed ucRNAs were identified using the Class Comparison Analysis of BRB tools version 3.6.0 (http://linus.nci.nih.gov/BRB-ArrayTools.html). The criterion for inclusion of a gene in the gene list was a p-value &lt;0.05. 
         [0075]    Real-time PCR analysis. RNA was extracted using Trizol reagent and treated with RNase-free DNase I (Qiagen, Valenica, Calif.). One μg of RNA was reverse transcribed to cDNA and quantitative real time PCR was performed using primers for uc.338 that spanned an intronic region, or primers for PCBP2 that spanned exonic regions distant to the location of uc.338, as follows: uc.338: 5′-AGCGACAGTGCGAGCTTT-3′, 3′-GGAAGGATTGAGTGAGCCTT-5′; PCBP2: 5′-TGACGCATGGCAACACC-3′, 5′-CGCCTTGACGCCCGATT-3′. Uc.338 and PCBP2 expression was normalized to that of RNU6B or GAPDH respectively. Genomic contamination was excluded by PCR of controls lacking reverse transcriptase for each sample. 
         [0076]    Cell fractionation. Cells (2×10 6 ) were pelleted and cytoplasmic and nuclear fractions obtained using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Sigma, St Louis, Mo.). RNA from each fraction was extracted using Trizol, and purity assessed using an Agilent bioanalyzer to detect the transfer RNA electropherogram band only in the cytosolic fractions as previously described (2). 
         [0077]    Transfection. Huh-7 cells were transfected using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif.) whereas HepG2 cells were transfected using the Nucleofector system, solution V program T28 (Amaxa Biosystems, Koln, Germany). siRNAs against uc.338 were designed using siDESIGN (http://www.dharmacon.com/sidesign), and the two highest ranked target sequences synthesized with sequences: siRNA-1 UGACAGCCCUGGAGACUGA and siRNA-2 CCACAGGACAGGUACAGCA. Cells were transfected with 100 nM siRNA to TUC338 or control (Dharmacon, Chicago, Ill.) for 48 hours before further experiments. siRNA against PCBP2 were purchased from Ambion (Ambion, Austin, Tex.). 
         [0078]    RACE. HepG2 RNA was used to generate RACE-ready cDNA by using the SMARTer RACE cDNA Amplification Kit (Clontech, Mountainview, Calif.) and following manufacturer&#39;s protocol. cDNA ends were amplified with Universal Primer Mix and gene-specific primers. Placental RNA and transferring receptor-specific primers provided by the manufacturer were used as controls. In case the primary PCT failed to give distint bands, we performed a “nested” PCR with the nested universal primer and the nested gene-specific primers. Primers were as follows (5′ to 3′): SI (5′ RACE), CAGTCTCGGCTGAGGCGGAAGGATTG; ASI (5′ RACE), GTGCCGGATTGACAGCCCTGGAGAC; SE (5′ RACE), CTCTAGAGGTGGTCCCTCCAGGTCAGG; nSI (5′ RACE), AAGGCTCACTCAATCCTTCC; nested ASI, TGACAGCCCTGGAGACTGAAATCCT; and antisense exonic, GGACAGGTACAGCACAGGCAGCGACAG. For the 3′ RACE RNA was polyadenylated by using the poly(A) polymerase TAK2180 (Takara). PCR products were then run in a 1.5% agarose gel, and DNA was extracted, cloned in TOPO.TA.2 plasmid, and sequenced with a 48-capillary Applied Biosystems 3730 DNA Analyzer. 
         [0079]    Western Blotting. HepG2 and Huh7 cells were serum-starved for 48 hours and then transfected as described above. After 48 hours, cells were collected and protein was extracted. Immunoblot analysis was performed as previously described (X). The primary anti-bodies were used at a concentration of 1:500 and were as follows: rabbit polyclonal anti-p16-INK4, mouse monoclonal anti-CDK4, and mouse monoclonal CDK6 (Cell Signaling); mouse monoclonal anti-cyclin D1 (BD Biosciences); and mouse monoclonal anti-proliferating cell nuclear antigen and rabbit polyclonal anti-vinculin (Santa Cruz Biotechnology, Santa Cruz, Calif.). 
         [0080]    Northern Blotting. For uc.338 detection, polyacrylamide gel-based Northern blotting was performed as previously described (4). The oligonucleotide probes used were complementary to the entire sequence of TUC338 or the entire cDNA of PCBP2. The size of the detected RNA was determined by using a size marker run on the same gel. 
         [0081]    uc.338 cloning. Genomic DNA was extracted from HepG2 cells using DNAzol (Invitrogen, Carlsbad, Calif.). The entire sequence of uc.338 was amplified using the following primers: uc.338-F: 5′-TTTTGTGTGAATTTCATGCTGGT-3′ and uc.338-R: 5′-GGTGTCCACAGCACCAAAAA-3′. The PCR product was sub-cloned into TOPO TA2.1 cloning vector (Invitrogen Carlsbad, Calif.), digested with Hind-III and Not-I (New England Biolabs, Ipswich, Mass.) and then cloned into the pSUPER.retro.neo+gfp vector (OligoEngine Seattle Wash.). The final product was verified by sequencing. Cells were transfected with 5 μg of uc.338-expressing or empty vector using lipofectamine and used after 48 hours. 
         [0082]    Gene annotation enrichment analysis. mRNA expression analysis was performed using Human Exon 1.0ST array (Affymetrix, Santa Clara, Calif.) in RNA obtained from HepG2 cells that were transfected with either control oligonucleotides or LNA-antisense to TUC338, which reduced uc.338 expression by 40% compared to controls by real-time PCR. Data were analyzed using the XRay software (Biotique Systems Inc, Reno, Nev.). Genes that were significantly differentially expressed were identified, and then compared to Gene Ontology classifications to identify over-representation in groups of the molecular function, cell processes or pathway classes. Microarray data has been deposited in the NCBI GEO repository. 
         [0083]    Cell growth assays. For anchorage-dependent growth, cells were plated (2×10 5 /well) in 6-well plates. Trypan blue staining was performed at each time point and the number of viable cells was expressed relative to cell counts at baseline. For anchorage-independent cell growth, cells were plated in 96-well plates (1000/well) in 0.6% agar containing medium with 20% FBS with 0.8% agar containing top and bottom feeder layers. Cell growth was fluorometrically assayed after 7 days using Alamar Blue (Biosource International, Camarillo, Calif.), and a FluoStar Omega Microplate Reader (BMG Labtech, Durham, N.C.). Final values were obtained by subtracting background fluorescence values from wells without cells. 
         [0084]    Cell cycle analysis. HepG2 cells were permeabilized with 75% ethanol and DNA stained using 50 μg/ml propidium iodide (PI), 0.1 mg/ml RNase A, 0.05% triton X-100 PBS. Cellular DNA content was measured by flow cytometry using a BD FACSCalibur (Heidelberg, Germany), and the proportions of cells in particular phases of the cell cycle were analyzed using the CellQuest Pro software. 
         [0085]    Tissue Microarray (TMA). A tissue microarray (TMA) was constructed from paraffin embedded-tissue samples obtained from 191 patients with HCC and adjacent non-tumoral tissue as previously described. Sections from the TMA block were used for in situ hybridization analysis. A separate commercially available TMA with 30 cases of HCC spotted in duplicate was also analyzed (Accumax array, IsuAbxis, Seoul, Korea). 
         [0086]    In situ RNA hybridization (ISH). A locked nucleic acid (LNA) probe with complementarity to a 22 bp section of uc.338 was labeled with 5′-digoxigenin and synthesized by Exiqon (Woburn, Mass., USA). Tissue sections on the TMA were digested using 2 mg/mL pepsin and ISH performed as described. Negative controls included omission of the probe and the use of a scrambled LNA probe. Each sample was classified by two independent reviewers based on the percentage of cells with detectable uc.338 expression as follows: negative (&lt;5%), weak (5-19%), moderate (20-49%) or strong (≧50%). An expression score was derived as the difference in percentage of cells that expressed uc.338 in HCC and in the corresponding adjacent liver, divided by the SD of % uc.338 expression across all samples analyzed. 
         [0087]    Statistical analysis. Results are expressed as mean±SEM, unless indicated otherwise. Comparisons between groups were performed using the two-tailed Student&#39;s t test. Significance was accepted when p was less than 0.05. 
         [0088]    Aberrant expression of selected ucRNAs in malignant hepatocytes. Genome-wide expression profiling identified 56 ucRNAs, representing 11% of all ucRNAs analyzed, that were aberrantly and significantly (p&lt;0.05) expressed in malignant HepG2 cells compared to non-malignant human hepatocytes ( FIG. 1A ). Of these, 33 were increased by 1.3 to 6.9-fold, whereas 23 were decreased by 0.8 to 0.3-fold in malignant hepatocytes (see Table 2). Exonic ucRNAs were not selectively enriched in malignant cells and the proportion of exonic regions in aberrantly expressed ucRNA (29% exonic, 42% non-exonic) was similar to those of all ultraconserved regions ( FIG. 1B ). The greatest change was noted for uc.338, which was increased in expression by 6.9±0.9 fold (p=0.001), and we thus focused our attention on this RNA gene. 
         [0089]    uc.338 is increased in expression in HCC cell lines. In humans, uc.338 is an exonic ucRNA encoded within the gene PCBP2 in chromosome 12. uc.338 is comprised of 223 nucleotides, of which 93 nucleotides overlap with the coding sequence of PCBP2 ( FIG. 2A ). By real-time PCR, a striking increase in uc.338 expression by 2.2 to 5.1-fold was observed in several HCC cell lines compared to non-malignant human hepatocytes ( FIG. 2B ). We next determined uc.338 expression in a panel of human cancer cell lines, including biliary, pancreatic, colorectal, prostatic and breast cancers. In most cells, the expression of uc.338 was reduced or comparable to the expression in normal hepatocytes. Interestingly, all cholangiocarcinoma cells showed very low levels of uc.338 expression, suggesting that uc.338 may differentiate between primary liver cancers arising from different hepatic epithelia. Thus, uc.338 is increased in HCC cells and might be a promising marker for HCC. 
         [0090]    uc.338 expression is increased in human HCC tissues. We next studied uc.338 expression in HCC tissues by in situ hybridization (ISH). 221 HCC samples in two tissue microarrays were analyzed. The arrays included 169 cases of adjacent non-cancerous liver tissue, with cirrhosis present in 97 cases. uc.338 expression was classified based on the percentage of cells with detectable expression as follows: negative (&lt;5%), weak (5-19%), moderate (20-49%) or strong (≧50%). uc.338 expression was detected in 170 cases (77%), with a moderate to strong expression in 62% of these ( FIG. 4A ). The mean uc.338 expression was 4.0±1.5% in non-cirrhotic liver, 15.0±4.5% in cirrhotic liver and 24.0±5.7% in HCC tissues ( FIG. 4B ). Adequate paired tumoral and adjacent non-tumoral tissue for analysis was available from 156 cases. Of these, uc.338 expression was increased in 62%, was unchanged in 24% or was decreased in 14% of HCC compared to adjacent tissues ( FIG. 4C ). Consistent increases in uc.338 expression (score&gt;2.0) were noted with HCC although a reduction in uc.338 expression (score&lt;−2.0) occurred sporadically ( FIG. 4D ). Within tumor cells, uc.338 expression was predominantly nuclear ( FIG. 5A ). Similarly, the nuclear/cytoplasmic ratio of uc.338 expression in HepG2 and Huh-7 cells was 17 and 27 respectively ( FIG. 5B ). 
         [0091]    uc.338 expression is regulated independently of PCBP2. uc.338 consists of 223 nt that are highly conserved throughout the species. In humans, the uc.388 ultraconserved region is located partly within the exon on the PCBP2 gene on chromosome 12. To evaluate the potential inter-relationships between PCBP2 and uc.338 transcription, we first examined the expression of PCBP2 by real time PCR in normal and HCC cell lines. The primers used spanned a genomic region in exons 10-13 of PCBP2 that was distant from that of uc.338 ( FIG. 2A ). PCBP2 expression was not increased in any of the HCC cell lines with the exception of Huh-7 cells, and PCBP2 expression did not correlate with that of uc.338 in all samples tested with a correlation coefficient of linear regression (R 2 ) of 0.18 ( FIG. 6A ). Thus, uc.338 expression occurs independently of PCBP2 despite their overlapping genomic locations. uc.338 expression was unchanged in HepG2 cells transfected with siRNA against PCBP2 despite an 85% reduction in PCBP2 mRNA expression. ( FIG. 6B ). We next evaluated the effect of uc.338 on PCBP2 expression in HepG2 and Huh-7 cells but did not observe any effect of reduction in uc.338 expression on PCBP2 expression ( FIG. 6C ). Two siRNAs were tested. siRNA-1 reduced uc.338 expression by 46% in HepG2 and 40% in Huh-7 cells whereas siRNA-2 reduced uc.338 expression by 54% in HepG2 and 45% in Huh-7 cells compared to scrambled nucleotide control siRNA. Moreover, transfection of normal human hepatocytes with a vector expressing full-length uc.338 resulted in a 1.4-fold increase in PCBP2 expression despite a 32-fold increase in uc.338 expression. These observations confirm that PCBP2 expression is not functionally modulated by uc.338 expression. 
         [0092]    Identification of Transcript Encoding uc.338. Having shown that uc.338 is transcribed independently of PCBP2, we proceeded to clone the transcript encoding this ultraconserved element. Rapid Amplification of cDNA Ends (“RACE”) was performed to characterize the 5′end and the 3′end of this transcript, which we termed “TUC338”. HepG2 RNA was retrotranscribed with the SMARTerScribeRT that exhibits terminal transferase activity upon reaching the end of an RNA template and adds residues to the first strand cDNA. The SMARTer oligo contains a terminal stretch of modified bases that anneal to the extended cDNA tail, allowing the oligo to serve as a template for the RT. Thus, a complete cDNA copy of the original RNA with an additional SMARTer sequence at the end is generated. To study TUC338 we used intronic primers that would not recognize PCBP2 coding sequence ( FIG. 7 ). No products were produced with the antisense intronic (ASI) primer suggesting that TUC338 is not encoded in antisense. Conversely, a few bands were produced after amplification with sense intronic (SI) primer, with the larger bands being around 500 nt. A nested PCR with the nested sense intronic (nSI) primer produced a single defined band of around 500 nt that was further sequenced, leading to the characterization of the 5′end of TUC338. As control, we used the sense exonic (SE) primer that produced a &gt;800-nt band by recognizing coding sequence PCBP2 ( FIG. 8 ). These findings further showed that the TUC338 transcript is different from the PCBP2 transcript and characterized the 5′end of TUC338. The 3′ RACE studies identified 130 nucleotides at the 3′end downstream of the ultraconserved sequence ( FIG. 9 ). In conclusion the uc.338 ultraconserved element is part of a 590 nt-long transcript, TUC388, that is transcribed independently on PCBP2 ( FIG. 10 ). 
         [0093]    Functional expression analysis of TUC338 regulated genes. To gain insight into the functional role of TUC338, we performed gene annotation enrichment analysis of genome-wide mRNAs that were changed in expression after TUC338 inhibition using siRNA. Functional annotation analysis identified the top four significantly over-represented cellular process gene classifications (and number of genes) as transcription (569), cell cycle (248), ubiquitin cycle (225) and cell division (115), whereas the top four over-represented molecular function classifications were ligase activity (159), protein binding (1,810), nucleotide binding (774), and ATP binding (638). The top four significantly over-represented GenMAPP pathway gene classifications were cell cycle-KEGG (56), mRNA processing reactome (63), RNA transcription reactome (28), and G1 to S cell-cycle reactome (41). These data suggested that TUC338 could modulate cellular processes involved in cell growth. 
         [0094]    TUC338 modulates cell growth in human hepatocytes. We assessed anchorage-dependent cell growth after transfection with either siRNA to TUC338 or scrambled nucleotide control siRNA. Compared to control siRNA, siRNA-1 and siRNA-2 reduced cell proliferation by 21%, (p&lt;0.001), or 24% (p=0.01) respectively, after 72 hours in HepG2 cells. Similar findings were observed in Huh-7 cells ( FIG. 11A ). Compared to controls there was a reduction of cells in S phase (p&lt;0.0001) in cells transfected with siRNA to TUC338 ( FIG. 11B ). Cancer is characterized by the acquisition of cellular traits that enhance cell growth under adverse micro-environmental conditions. Therefore, we examined anchorage-independent growth by examining growth in soft agar assays. Compared to controls, siRNA to TUC338 reduced soft agar growth of HepG2 cells by 40.0±2.0% ( FIG. 11C ). 
         [0095]    TUC338 modulates cell growth in mouse hepatocytes. The sequence conservation of ucRNAs across diverse species suggests that these genes may participate in essential roles that may be similar across species. To examine cross-species similarities in the effects of TUC338, we studied the effect of this gene in modulating transformed cell growth in murine cells. First, we examined the effect of cell transformation on TUC338 expression in BNL-CL.2 embryonic mouse hepatocytes. Compared to the parental BNL-CL.2 cells, the expression of TUC338 was increased by 2.1-fold in BNL-SVA.8 cells which are derived from BNL-CL.2 by SV40 transformation ( FIG. 12A ). BNL-SVA.8 cells have increased anchorage-dependent and anchorage-independent growth in soft agar compared to BNL-CL.2 cells ( FIGS. 12 , B &amp; C). Knockdown of TUC338 in BNL-SVA.8 cells using siRNA caused a dramatic reduction in cell proliferation. At 72 hours, cell proliferation was reduced by 65.0±2.0% (p=0.0001) in BNL-SVA.8 cells transfected with siRNA to TUC338 compared to control siRNA ( FIG. 12D ). These data, indicating a relationship among TUC338 expression, cell transformation, and cell growth in mouse hepatocytes, are similar to those observed in humans. 
         [0096]    TUC338 modulates progression through the cell cycle. The functional genomic expression analysis showed enrichment in genes involved in cell cycle progression from phase G1 to phase S in response to inhibition of TUC338. Moreover, inhibition of TUC338 in HCC reduced the number of cells in S phase ( FIG. 11 ). Thus, we assessed the effect of TUC338 inhibition on expression of several proteins involved in the G1/S checkpoint in HepG2 and Huh7 cells. S phase progression can be modulated by CDK4/6-cyclin D1 mechanisms, and alterations in cyclin D are prominent in HCC. After inhibition of TUC338, we observed an increase in expression of the tumor suppressor p16INK4a and an associated reduction of CDK4, CDK6 and cyclin D1 ( FIG. 13 ). Indeed, altered expression of these cell cycle regulatory proteins are associated with HCC growth: Although S-phase progression can also be modulated by the CDK2-cyclin E, effects on cell growth after inhibition of TUC338 were noted in Huh7 cells, which do not express p21 and cyclin E, making it unlikely that the effects of TUC338 on cell cycle progression involved these proteins. 
         [0097]    Testing for uc.338 in human blood samples. It should be recognized that the above experiments can be done using blood samples. In such a circumstance, serum samples are obtained from individuals with hepatocellular cancer (HCC) or chronic liver disease without HCC. 50 fmol mmu-miR-295 mimics (Qiagen, Valencia, Calif.) are added into 100 μl serum and incubated for 5 minutes. RNA is then extracted using TRIZOL reagent (Invitrogen, Carlsbad, Calif.). Briefly, 1.0-mi TRIZOL reagent and 200-μl chloroform are added to the serum sample and the mixture is vortexed for 15 seconds and kept at 25° C. for 3 minutes. After centrifugation at 12,000 g for 15 minutes at 4° C., the supernatant is transferred to a fresh tube and 500-μl isopropanol is added. After incubation at −20° C. for 20 minutes, the mixture is centrifuged at 12,000 g for 10 minutes at 4° C. to remove the supernatant and the RNA pellet is washed with 75% ethanol. After removal of ethanol by centrifugation at 7500 g for 5 min at 4° C., RNA is air-dried for 5 minutes and then dissolved in 30-μl RNase-free water. Each sample of 11.5-μl RNA is polyadenylated and reversely transcribed to cDNA in a final volume of 30 μl using polyadenylation polymerase (New England Biolabs, Beverly, Mass.) and First-Strand cDNA Synthesis Kit with oligo-d(T) primer. The cDNA product is 1:5 diluted with water and stored at −80° C. for analysis. Real-time quantitative PCR (qPCR) quantification of TUC338 is performed using SYBR Green PCR Master Mixture. mmu-miR-295 is used as an internal normalization control. Melting curve analysis is performed at the end of PCR cycles in order to validate the specificity of the expected PCR product. All samples are run in duplicate, including blank controls without cDNA. The cycle threshold (Ct) is defined as the number of cycles required for the fluorescent signal to cross the threshold in qPCR. The formula 2 ΔCt  is used to calculate the levels of TUC338 in serum, where ΔCt=mean (Ct of mmu-miR295)-Ct of TUC338. 
       OTHER EMBODIMENTS 
       [0098]    It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.