Abstract:
The present invention relates to use of two microRNAs in detection of lung cancer prognosis and in medicine preparation. Particularly, the invention relates to a composition comprising two small RNA molecules microRNA-150 and microRNA-886-3p, a device comprising the composition used in detection of lung cancer prognosis and in preparation of medicaments for inhibiting mammal and human lung cancer metastasis. Specifically, the expression levels of microRNA-150 and microRNA-886-3p can be used as the prognostic criteria of lung cancer prognosis, wherein high expression level of the gene combination indicates favorable therapeutic effect. The invention also relates to a device detecting the expression levels of microRNA-150 and microRNA-886-3p in mammalian and human lung cancer and a method for detecting the expression levels of microRNA-150 and microRNA-886-3p in samples.

Description:
PRIORITY CLAIM TO RELATED APPLICATIONS 
     This application is a national stage application under 35 U.S.C. §371 of PCT/CN2009/072695, filed Jul. 9, 2009, and published as WO 2011/003237 A1 on Jan. 13, 2011, which application and publication are incorporated by reference as if reproduced herein and made a part hereof in their entirety, and the benefit of priority of is claimed herein. 
     TECHNICAL FIELD 
     The invention relates to a composition including two microRNA molecules microRNA-150 and microRNA-886-3p, a device containing the composition for detecting prognosis of lung cancer and a use of the composition in the preparation of medicaments for inhibiting mammalian and human lung cancer transformation. Specifically, the expression level of microRNA-150 and microRNA-886-3p can be used as a prognostic marker in lung cancer, wherein, high expression of the gene combination in the test sample indicates good therapeutic effect. The invention also relates to a device for detecting the expression level of microRNA-150 and microRNA-886-3p in mammalian and human lung cancer patients and a method for detecting the expression level of microRNA-150 and microRNA-886-3p in the samples to be tested. 
     BACKGROUND OF THE INVENTION 
     Molecular biology techniques can be employed for an effective detection at molecular level to predict prognosis in patients with malignant tumor, and then, for a proper individual treatment of these diseases. Lung cancer is a common cancer worldwide, the incidence rate in male is 18%, while 21% in female. In 2005, approximate 500,000 new cases of lung cancer occurred in China (about 330,000 cases for male and 170,000 cases for female). The mortality of lung cancer is high, over 900,000 patients die from lung cancer each year (Parkin D M 1999). Therein, 15-20% of lung cancer cases are due to small cell lung cancer. Compared to non small cell lung cancer, small cell lung cancer is of unique morpha, substructure, immunohistochemical features and is classified as a kind of neuroendocrine tumors (Morita T 1990). The disease progresses rapidly, meanwhile, it is sensitive to the first course of radiotherapy and chemotherapy and the response rate is up to 60-80%, but it relapses soon after the treatment, and then becomes resistant to radiotherapy and chemotherapy. Only the patients who have small cell lung cancer at 15-25% limited stage and less than 5% extensive stage survive another five years via the treatment (Sandler A B 2003). In addition, 25-40% of the patients having small cell lung cancer are more than 70 years old at diagnosis. The patients poorly tolerate the chemotherapy because of complications and the like, and are badly prognosticated due to the limited treatment means, so their median survival time is only 10 months (Sekine I 2004). Therefore, there is an urgent need to develop a new treatment strategy which can improve the prognosis of small cell lung cancer. 
     Molecular targeted therapy has been a hot research point in recent years, and has made a breakthrough in treatment of some malignant tumors. For example, Gefinitib (Iressa) was used in non small cell lung cancer treatment, and the prognosis was good, especially for those patients who are female, do not smoke and suffer from adenocarcinoma; and Imatinib was used in treatment of gastrointestinal stromal tumors, and the better therapeutic effect had been obtained especially for those with Kit exon 11 mutations (Nilsson B 2007); and a combination of C225 and radiotherapy was employed in treatment of locally advanced head and neck cancer, and the survival rate was increased nearly 1 time than that of using radiotherapy alone (Bonner J A 2006). Therefore, it will be a great help for improving patient&#39;s prognosis to further understand the molecular mechanism of small cell lung cancer. Fischer et al. (Fischer B 2007) summarized the molecular mechanism studies of small cell lung cancer in recent 20 years: the molecular pathways involved in small cell lung cancer consist of mainly two pathways, PI3K/Akt/mTOR and RAS/MAPK, which are activated through binding of the cell surface receptor tyrosine kinases (RTKs) and their corresponding extracellular growth factors, wherein the RTKs mainly include IGF-IR, EGFR, VEGFR, PDGFR, c-MET. Thus, theoretically, inhibition of the growth of small cell lung cancer can be achieved through inhibition of RTKs or key targets in the pathways. However, it is a pity that no desired clinical effect has been obtained yet. In view of this, it may be a breakthrough for treatment to understand other aspects of the molecular mechanisms in small cell lung cancer. 
     Small RNA molecule (MicroRNA) generally consists of 18-25 nucleotides, which is a non-coding RNA molecule and can inhibit mRNA function and regulate translation process by binding to said target mRNA. Since 2005, a small amount of literatures on the relationship of microRNA and prognosis have been published, and have confirmed in chronic lymphoma, acute myeloid leukemia, non-small cell lung cancer, pancreatic cancer and neuroblastoma, colon cancer, that prognosis is significantly influenced by microRNA. However, studies on the effects of microRNA on the prognosis of small cell lung cancer have not yet been reported. 
     MicroRNA-150 (miR-150 for short), containing 22 nucleotides, locates on chromosome 19 and its sequence is shown as SEQ ID NO. 1: 5′-UCUCCCAACCCUUGUACCAGUG-3′, with GenBank accession No. NT — 011109.15, (sequence 22272232˜22272315), which is commonly expressed in mature lymphocytes. As reported by Xiao C in 2007, the main function of microRNA-150 is to control the growth and differentiation of B lymphocytes by regulating c-Myb transcription factor. MicroRNA-886-3p (miR-886-3p for short), containing 21 nucleotides, locates on chromosome 5, and its sequence is shown as SEQ ID No. 2: 5′-CGCGGGUGCUUACUGACCCUU-3′, with GenBank accession No. NT — 034772.5 (sequence 3783,1310˜3783,1190), the function of which has not been reported in the literature. 
     SUMMARY OF THE INVENTION 
     Therefore, the first aspect of the present invention relates to a composition comprising a therapeutically effective amount of two microRNA molecules of microRNA-150 and microRNA-886-3p, wherein the sequences of microRNA-150 and microRNA-886-3P are shown as SEQ ID NO. 1: 5′-UCUCCCAACCCUUGUACCAGUG-3′ and SEQ ID NO. 2: 5′-CGCGGGUGCUUACUGACCCUU-3′, respectively. More particularly, the composition also includes a preservative for prevention of the degradation of microRNA molecules and a pharmaceutically acceptable carrier. 
     The second aspect of the present invention relates to a device containing the composition as described in the first aspect used for the detection of lung cancer prognosis. Specifically, the expression level of microRNAs, microRNA-150 and microRNA-886-3p, can be used as a criteria in the prognosis of lung cancer, in that high expression of the two microRNAs indicates a good prognosis for patients. More particularly, said lung cancer is small cell lung cancer. More particularly, said device is a gene chip or a reagent kit. 
     The third aspect of the present invention relates to use of the composition as described in the first aspect in preparation of medicaments for inhibiting mammalian and human lung cancer transformation. Particularly, said lung cancer is small cell lung cancer. 
     The fourth aspect of the present invention relates to a reagent kit for detecting expression status of microRNAs of microRNA-150 and microRNA-886-3P in mammalian and human lung cancer patients, comprising the followings: 
     1) Optionally, the reagents used for extracting microRNA from the patients, 
     2) SEQ ID NO.3, used as a primer of microRNA reverse transcription: 
     
       
         
               
               
             
           
               
                   
                 5′-GTGCAGGGTCCGAGGT-3′, 
               
             
          
         
       
     
     3) a universal sense primer of microRNA: 
     
       
         
               
             
           
               
                 SEQ ID NO. 4: 
               
               
                 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACC 
               
               
                   
               
               
                 ACTGG-3′, 
               
               
                 (for microRNA-150) 
               
               
                   
               
               
                 SEQ ID NO. 5: 
               
               
                 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACA 
               
               
                   
               
               
                 AGGGT-3′, 
               
               
                 (for microRNA-886-3p) 
               
             
          
         
       
     
     4) a specific antisense primer of microRNA 
     
       
         
               
               
             
           
               
                   
                 SEQ ID NO.6: 
               
               
                   
                 5′-GTCTCCCAACCCTTGTACCA-3′, 
               
               
                   
                 (for microRNA-150) 
               
               
                   
                   
               
               
                   
                 SEQ ID NO.7: 
               
               
                   
                 5′-CACGCGGGTGCTTACTGAC-3′, 
               
               
                   
                 (for microRNA-886-3p) 
               
             
          
         
       
     
     5) Optionally, other necessary reagents used for reverse transcription PCR or PCR, and 
     6) Optionally, other reagents used for detection of said microRNA, reverse transcription PCR products or PCR products, 
     wherein the sequences of microRNA-150 and microRNA-886-3P are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2. Particularly, said device is a gene chip or a reagent kit. 
     The fifth aspect of the present invention relates to a method for detecting expression status of microRNA-150 and microRNA-886-3p in the samples to be tested, comprising the steps of: 
     1) Extracting microRNA from freshly isolated tissues or formalin-paraffin-embedded tissues, 
     2) Detecting tumor tissue specimens by means of gene chips, and screening genes associated with prognosis: microRNA-150 and microRNA-886-3p, 
     3) Processing the data from chips, comprising: 
     Evaluating the relationship between each microRNA signal expression value and survival rate by means of univariate Cox regression model; 
     Assigning each patient a compound value, via a linear combination of the statistically significant signal values of the microRNAs multiplied by the regression coefficients derived from the univariate Cox regression analyses; 
     Evaluating prognosis, using the median compound value as the cut-off point, with the formula of:
 
Compound value=0.545×(expression value of microRNA-150)+0.617×(expression value of microRNA-886-3 p ),
 
     4) Verifying the prognostic model, wherein PCR amplification is performed with primers designed according to microRNA-150 and microRNA-886-3p gene sequences, and 2˜4 μl of PCR products is detected by 1.5% non-denaturing agarose gel electrophoresis (without formaldehyde), 
     5) Collecting and processing PCR data: 
     Data are normalized using U6 RNA as an internal standard, and the prognosis is evaluated with the model built with chips. 
     In other words, the inventor studies on lung cancer, especially small cell lung cancer in China by means of microRNA gene chips and qRT-PCR techniques. Unexpectedly, the expression levels of microRNA-150 and microRNA-883-3p are different in the tissue samples from patients with different prognosis. Most patients with good prognosis have high expression of the two microRNAs. It is particularly important that after a linear combination of the signal values of the two microRNAs, the expression is more closely related to prognosis. Detection was carried out in another set of samples and similar results was obtained. Therefore, a combination of the genes can be used as a good prognostic model for lung cancer, especially for small cell lung cancer. With this model, lung cancer can be classified into two types, indolent and invasive, and different therapeutical regimens which will be employed depend on different types. Indolent lesions can be treated by using local treatment such as surgery, radiotherapy, and the treatment is relatively more aggressive for invasive lesions, mainly through systemic chemotherapy. By applying the gene combination to a corresponding gene chip or reagent kit, the prognosis can be rapidly known for lung cancer, especially for small cell lung cancer, in mammals including human beings, which will be of epoch-making significance in changing the therapeutical mode of lung cancer, especially small cell lung cancer. 
     In one embodiment of the invention, the effect of a composition containing microRNA-150 and microRNA-886-3p on the in vitro invasion and adhesion ability of lung cancer cell lines is described. The results show that microRNA-150 and microRNA-886-3p can diminish adhesive capacity of lung cancer cell to extracellular matrix and inhibit the invasive metastasis of lung cancer cells. Therefore, the microRNA molecules of microRNA-150 and microRNA-886-3p as mentioned in the invention have a potential in use for the treatment of lung cancer, especially small cell lung cancer. 
     In another embodiment of the invention, the detection method according to the present invention is described, which mainly comprises the steps of microRNA extraction, gene chip preparation, hybridization, and qRT-PCR verification and the like. Gene chip detection is mainly used for screening genes associated with prognosis. The genes which are screened out are verified by qRT-PCR. Since the inventor has found the genes associated with the prognosis of small cell lung cancer, it is feasible to use only microRNA extraction and qRT-PCR in future clinical applications. These two methods are conventional operations for those skilled in the art. Therefore, it is easy to clinically popularize the model. 
     In another embodiment of the invention, a method for detecting the expression status of microRNA-150 and microRNA-886-3p in the samples to be tested is also described, which comprises the following steps of: 
     1) Extracting microRNA from freshly isolated tissues or formalin paraffin embedded tissues, 
     2) Detecting tumor tissue specimens by means of gene chips, and screening genes associated with prognosis: microRNA-150 and microRNA-886-3p, 
     3) Processing the data from chips, comprising: 
     evaluating the relationship between each microRNA signal expression value and survival rate by means of univariate Cox regression model; 
     Assigning each patient a compound value, via a linear combination of the statistically significant signal values of the microRNAs multiplied by the regression coefficients derived from the univariate Cox regression analyses; 
     Evaluating prognosis, using the median compound value as the cut-off point, with the formula of:
 
Compound value=0.545×(expression value of microRNA-150)+0.617×(expression value of microRNA-886-3 p ),
 
     4) Verifying the prognostic model, wherein PCR amplification is performed with primers designed according to microRNA-150 and microRNA-886-3p gene sequences, and 2˜4 μl of PCR products is detected by 1.5% non-denaturing agarose gel electrophoresis (without formaldehyde), and 
     5) Collecting and processing PCR data: 
     Data are normalized using U6 RNA as an internal standard, and the prognosis is evaluated with the model built with chips. 
    
    
     
       DETAILED DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows the electrophoresis results of microRNA-150 and microRNA-886-3p from small cell lung cancer tissues, wherein 
       1˜40 hsa-miR-let-7i gene is amplified by RealTime PCR, respectively using 1st-cDNA of sample 145, 146, 147, 148, 151, 152, 156, 157, 158, 160, 162, 163, 165, 167, 168, 169, 170, 172, 174, 176, 177, 178, 179, 180, 181, 182, 183, 184, 186, 188, 189, 192, 195, 195, 196, 197, 198, 199, 200 and 202 as the template; 
       41˜80 hsa-miR-150 gene is amplified by RealTime PCR, respectively using 1st-cDNA of sample 145, 146, 147, 148, 151, 152, 156, 157, 158, 160, 162, 163, 165, 167, 168, 169, 170, 172, 174, 176, 177, 178, 179, 180, 181, 182, 183, 184, 186, 188, 189, 192, 195, 195, 196, 197, 198, 199, 200 and 202 as the template; 
       81˜120 hsa-miR-886-3p gene is amplified by RealTime PCR, respectively using 1st-cDNA of sample 145, 146, 147, 148, 151, 152, 156, 157, 158, 160, 162, 163, 165, 167, 168, 169, 170, 172, 174, 176, 177, 178, 179, 180, 181, 182, 183, 184, 186, 188, 189, 192, 195, 195, 196, 197, 198, 199, 200 and 202 as the template. 
       Molecular weight marker: TaKaRa DL2000, sizes of marker DNAs comprising 100 bp, 250 bp, 500 bp, 750 bp, 1000 bp, 2000 bp (from bottom to top) 
       As indicated in the results of electrophoresis, there is a good specificity in the microRNA RealTime PCR reaction. 
         FIG. 2  shows that the prognosis of patients with small cell lung cancer in the chip group is analyzed according to the expression status of the prognostic model. 
         FIG. 3  shows that the prognosis of patients with small cell lung cancer in the PCR Verification group is analyzed according to the expression status of the prognostic model. 
         FIG. 4  shows the verification of RNA extraction in table 1. 
         FIG. 5  shows that microRNA-886-3p and microRNA-150 inhibit the in vitro invasion ability of the cells, 
       A: The schematic diagram of cell staining shows the inhibition of in vitro invasion ability of H446 cells after miR control, miR-150, miR-886-3p, miR-150/miR-886-3p was respectively added to the cell line, 
       B: The histogram shows the inhibition of in vitro invasion ability of H446 cells after miR control, miR-150, miR-886-3p, miR-150/miR-886-3p was respectively added to the cell line. 
         FIG. 6  shows that microRNA-886-3p and microRNA-150 inhibit the in vitro invasion ability of the cells, 
       A: The schematic diagram of cell staining shows the inhibition of in vitro invasion ability of H1299 cells after miR control, miR-150, miR-886-3p, miR-150/miR-886-3p was respectively added to the cell line, 
       B: The histogram shows the inhibition of in vitro invasion ability of H1299 cells after miR control, miR-150, miR-886-3p, miR-150/miR-886-3p was respectively added to the cell line. 
         FIG. 7  shows that microRNA-886-3p and microRNA-150 inhibit the cell adhesion to matrix, 
       A: The histogram shows the adhesion rate of H446 cells at 30, 60, 90 minutes post miR-NC (control), miR-886-3p, miR-150, miR-886-3p/miR-150 transfection, 
       B: The histogram shows the adhesion rate of H1299 cells at 30, 90 minutes post miR-NC (control), miR-886-3p, miR-150, miR-886-3p/miR-150 transfection. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention will be further described by use of the following examples, but not limited to these ones. 
     In the following examples, unless specifically indicated, all the reagents used in the application are analytically pure and commercially available. Unless otherwise indicated, RT-PCR, PCR and other operations as mentioned in the examples of the invention are performed in accordance with “Molecular Cloning: a Laboratory Manual (The 3rd Edition)” (J. Sambrook and D. W. Russell [USA], translated by Peitang Huang et al, Science Press, 2002) and the manufacturer&#39;s instruction; cell culture, cell passage, cell recovery and cryopreservation, cell transfection, immunofluorescence assay and other operations are carried out in accordance with “Culture of Animal Cells: a Manual of Basic Technique (The 4th Edition)” (R. Ian Freshney [UK], translated by Jingbo Zhang et al, Science Press, 2000) and the manufacturer&#39;s instruction. 
     Example 1 
     The Method for Detecting Gene Expression Status of MicroRNA-150 and MicroRNA-886-3p 
     1. Extraction of Total RNA of Samples—Extraction of Total RNA from Tissues or Cells with Trizol Method 
     (1) Sample Source 
     Samples were collected from the Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College. The inclusion criteria comprises: Age≦75; KPS scores≧80; The patients with small cell lung cancer in limited stage, receiving surgery±chemotherapy±radiotherapy; Sufficient formalin-paraffin-embedded tissues to obtain enough miRNA; Complete written medical records and follow-up records. 42 cases of formalin-fixed and paraffin-embedded small cell lung cancer specimens between 2002 and 2005 which met the inclusion criteria were chosen for the microRNA chip detection and screening of microRNAs associated with prognosis. 40 cases of formalin-fixed and paraffin-embedded small cell lung cancer specimens between 2000 and 2001 were chosen for verification of the chip results. The characteristics of specific cases are shown in table 1. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 The characteristics of specific cases 
               
             
          
           
               
                   
                   
                 Chip  
                 Verification 
                   
               
               
                   
                   
                 group 
                 group 
                 P  
               
               
                   
                 Variable 
                 N = 42(%) 
                 N = 40(%) 
                 value 
               
               
                   
               
             
          
           
               
                 Age(year) 
                 Mean ± SD 
                 60.1 ± 11.1 
                 54.3 ± 11.8 
                 0.025 
               
               
                   
                 Range 
                 33~74 
                 33~73 
                   
               
               
                 Gender 
                 Male 
                 30(71.4) 
                 32(80.0) 
                   
               
               
                   
                 Female 
                 12(28.6) 
                  8(20.0) 
                 0.445 
               
               
                 Smoking 
                 Yes 
                 30(71.4) 
                 29(72.5) 
                   
               
               
                   
                 No 
                 12(28.6) 
                 11(27.5) 
                 1.000 
               
               
                 Stage 
                 I 
                 16(38.1) 
                  6(15.0) 
                   
               
               
                   
                 II 
                 14(33.3) 
                 10(25.0) 
                 0.010 
               
               
                   
                 III 
                 12(28.6) 
                 24(60.0) 
                   
               
               
                 Tumor 
                 Superior lobe 
                 25(59.5) 
                 21(52.5) 
                 0.657 
               
               
                 location 
                 Intermediate and  
                 17(40.5) 
                 19(47.5) 
                   
               
               
                   
                 inferior lobe 
                   
                   
                   
               
               
                 Treatment 
                 Surgery + chemotherapy 
                 39(92.9) 
                 32(80) 
                   
               
               
                   
                 Surgery + chemotherapy +  
                  2(4.8) 
                  7(17.5) 
                 0.120 
               
               
                   
                 radiotherapy 
                   
                   
                   
               
               
                   
                 Surgery alone 
                  1(2.4) 
                  1(2.5) 
               
               
                   
               
             
          
         
       
     
     (2) Sample Processing (Grinding is not Necessary for Bacteria or Cells) 
     The tissue sample with an area of about 1 cm 2  was broken in aluminum foil, then transferred to an Eppendorf tube containing steel beads and ground with a grinding mill (30 l/s, 8 min), 
     note: This step should be operated as much as possible in a cryogenic liquid nitrogen environment, and grinding is not necessary for bacteria or cells; 
     (3) 1 ml of trizol was added into the Eppendorf tube after the grinding step, and mixed by shaking; 
     (4) The mixed solution was transferred to a new Eppendorf tube, and 200 μl of chloroform was added and mixed by shaking; 
     (5) The solution was centrifuged at 12000 rpm for 15 min at 4° C. 
     (6) The obtained supernatant was transferred to a new Eppendorf tube, and 500 μl of isopropanol was added, then gently mixed and placed for 15 min at room temperature; 
     (7) The solution was centrifuged at 12000 rpm for 15 min at 4° C. 
     (8) The supernatant was removed, and then 1 ml of 75% ethanol was added and mixed by shaking; 
     (9) The solution was centrifuged at 7500 rpm for 5 min at 4° Q 
     (10) The supernatant was removed, and the ethanol was totally evaporated in a laminar flow cabinet; 
     (11) 40˜60 μl of DEPC H 2 O was added, and the mixture was solubilized at 65° C. for 5 min; 
     (12) The obtained sample was frozen at −20° C. for cryopreservation. 
     2. Quality Assessment of Total RNA 
     (1) Determination of total RNA concentration by Nanoprop (loading 2 μl of total RNA), 
     (2) Determination of RNA quality by means of 1.5% formaldehyde denaturing agarose gel electrophoresis 
     
       
         
               
               
               
               
             
               
               
             
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Total RNA 
                   
                 500 ng 
               
               
                   
                 5 × Loading Buffer 
                 μl 
                 2 
               
               
                   
                 DEPC H 2 O 
                   
                 to 8~9 μl 
               
             
          
           
               
                   
                 The mixture was denatured at 65° C. for 5 min,  
               
               
                   
                 then put into an ice bath for 5 min 
               
             
          
           
               
                   
                 EB(500-fold dilution) 
                   
                 1 μl 
               
               
                   
                   
               
               
                   
                 The total volume is about 6~8 μl. 
               
             
          
         
       
     
     Formaldehyde denaturing agarose gel: 0.45 g of agarose was added into 30 ml of 1×TBE Buffer, the mixture was heated to melt in a microwave oven and gently shaken to mix the agarose thoroughly (no suspending granules can be visually observed), 600 μl of formaldehyde was added when the mixture cooled to about 60° C., mixed, and then poured into a special gel casting module for RNA (7.5×5.5 cm). After being placed for about 30 min at room temperature, the agarose gel would be ready to use. 
     The electrophoresis was performed at 120˜130V for 15˜20 min 
     The quantity, degradation and size of the RNAs extracted from the 40 specimens in the invention are shown in table 2 and  FIG. 4 . 
     
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 The vertification of RNA 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                 Total 
                   
               
               
                   
                   
                   
                   
                   
                 Concentration 
                 amount 
                 Results of 
               
               
                 Lane 
                 A 260   
                 A 280   
                 A 260/280   
                 A 260/230   
                 (ng/μl) 
                 (μg) 
                 electrophoresis 
               
               
                   
               
             
          
           
               
                 1 
                 62.489 
                 34.048 
                 1.84 
                 2.26 
                 2499.56 
                 499.912 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 2 
                 8.054 
                 4.766 
                 1.69 
                 1.93 
                 322.14 
                 64.428 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 3 
                 95.425 
                 52.686 
                 1.81 
                 2.17 
                 3817 
                 763.4 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 4 
                 65.457 
                 35.985 
                 1.82 
                 2.19 
                 2618.27 
                 523.654 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 5 
                 11.611 
                 6.536 
                 1.78 
                 2.18 
                 464.43 
                 92.886 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 6 
                 57.839 
                 31.169 
                 1.86 
                 2.05 
                 2313.57 
                 462.714 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 7 
                 113.089 
                 67.862 
                 1.67 
                 2.01 
                 4523.56 
                 904.712 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 8 
                 69.717 
                 37.33 
                 1.87 
                 2.21 
                 2788.69 
                 557.738 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 9 
                 31.285 
                 17.133 
                 1.83 
                 2.32 
                 1251.39 
                 250.278 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 10 
                 103.177 
                 57.997 
                 1.78 
                 2.17 
                 4127.07 
                 825.414 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 11 
                 83.719 
                 45.835 
                 1.83 
                 2.31 
                 3348.76 
                 669.752 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 12 
                 70.802 
                 38.252 
                 1.85 
                 2.21 
                 2832.08 
                 566.416 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 13 
                 41.937 
                 23.6 
                 1.78 
                 2.25 
                 1677.49 
                 335.498 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 14 
                 10.302 
                 5.742 
                 1.79 
                 1.97 
                 412.08 
                 82.416 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 15 
                 63.282 
                 35.251 
                 1.8 
                 2.3 
                 2531.29 
                 506.258 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 16 
                 9.169 
                 5.158 
                 1.78 
                 2.02 
                 366.75 
                 73.35 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 17 
                 33.184 
                 17.966 
                 1.85 
                 2.32 
                 1327.37 
                 265.474 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 18 
                 22.921 
                 12.538 
                 1.83 
                 2.4 
                 916.85 
                 183.37 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 19 
                 14.606 
                 8.118 
                 1.8 
                 2.36 
                 584.25 
                 116.85 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 20 
                 27.114 
                 14.844 
                 1.83 
                 2.14 
                 1084.56 
                 216.912 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 21 
                 33.802 
                 18.46 
                 1.83 
                 2.11 
                 1352.08 
                 270.416 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 22 
                 34.601 
                 18.898 
                 1.83 
                 2.29 
                 1384.05 
                 276.81 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 23 
                 0.25 
                 0.15 
                 1.66 
                 1.5 
                 9.98 
                 1.996 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 24 
                 10.171 
                 5.778 
                 1.76 
                 1.99 
                 406.84 
                 81.368 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 25 
                 48.25 
                 26.973 
                 1.79 
                 2.12 
                 1929.99 
                 385.998 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 26 
                 20.812 
                 11.576 
                 1.8 
                 2.14 
                 832.47 
                 166.494 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 27 
                 11.803 
                 6.701 
                 1.76 
                 2.23 
                 472.12 
                 94.424 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 28 
                 35.502 
                 19.186 
                 1.85 
                 2.05 
                 1420.09 
                 284.018 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 29 
                 48.24 
                 25.835 
                 1.87 
                 2.34 
                 1929.6 
                 385.92 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 30 
                 64.657 
                 34.963 
                 1.85 
                 2.29 
                 2586.28 
                 517.256 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 31 
                 16.224 
                 9.008 
                 1.8 
                 2.22 
                 648.96 
                 129.792 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 32 
                 29.56 
                 16.061 
                 1.84 
                 2.2 
                 1182.41 
                 236.482 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 33 
                 45.883 
                 24.671 
                 1.86 
                 2.21 
                 1835.34 
                 367.068 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 34 
                 27.885 
                 15.132 
                 1.84 
                 2.25 
                 1115.39 
                 223.078 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 35 
                 35.712 
                 18.743 
                 1.91 
                 2.14 
                 1428.47 
                 285.694 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 36 
                 13.813 
                 7.397 
                 1.87 
                 2.2 
                 552.5 
                 110.5 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 37 
                 44.185 
                 23.706 
                 1.86 
                 2.16 
                 1767.42 
                 353.484 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 38 
                 14.484 
                 7.836 
                 1.85 
                 1.94 
                 579.36 
                 115.872 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 39 
                 33.135 
                 18.072 
                 1.83 
                 2.12 
                 1325.41 
                 265.082 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                 40 
                 42.151 
                 23.146 
                 1.82 
                 2.24 
                 1686.04 
                 337.208 
                 RNA 
               
               
                   
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                   
               
             
          
         
       
     
     3. Reverse Transcription of MicroRNA: 
     (1) Reaction System of Reverse Transcription 
     
       
         
               
               
               
             
               
               
             
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Total RNA 
                 100 ng 
               
               
                   
                 Reverse transcription primer of  
                   1 μl(1 μM) 
               
               
                   
                 microRNA: SEQ ID NO. 
                   
               
               
                   
                 DEPC H 2 O 
                 to 12.3 μl 
               
             
          
           
               
                   
                 The mixture was denatured at 65° C. for 5 min,  
               
               
                   
                 then put into an ice bath for 5 min. 
               
             
          
           
               
                   
                 5 × 1 st  Buffer 
                   4 μl 
               
               
                   
                 0.1M DTT 
                   2 μl 
               
               
                   
                 dNTPs 
                 0.5 μl(10 mM for each) 
               
               
                   
                 RNase Inhibitor 
                 0.2 μl(40 U/μl) 
               
               
                   
                 M-MLV 
                   1 μl(200 U/μl) 
               
               
                   
                   
               
               
                   
                 The total volume of the system was 20 μl. 
               
             
          
         
       
     
     (2) The Program of Reverse Transcription was: 16° C. for 30 min, 37° C. for 30 min, 70° C. for 10 min, and then the Products were Kept at 4° C. to be Used. 
     4. Realtime PCR Reaction of MicroRNA 
     (1) Reaction System of MicroRNA RealTime PCR 
     
       
         
               
             
               
               
             
           
               
                   
               
               
                 Template(cDNA) 1 μl of the 20 μl reaction system  
               
               
                 of reverse transcription in general 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 MgCl 2   
                 1.6 μl 
               
               
                 Primers: universal sense primer of microRNA 
                 0.6 μl (10 μM) 
               
               
                 For microRNA-150: SEQ ID NO. 4 
                   
               
               
                 For microRNA-886-3p: SEQ ID NO. 5 
                   
               
               
                 Specific antisense primer 
                 0.6 μl (10 μM) 
               
               
                 For microRNA-150: SEQ ID NO. 6 
                   
               
               
                 For microRNA-886-3p: SEQ ID NO. 7 
                   
               
               
                 DNA Master SYBR Green I MIX 
                   2 μl 
               
               
                 Nuclease Free H 2 O was added to 20 μl 
               
               
                   
               
             
          
         
       
     
     (2) Program of MicroRNA RealTime PCR 
     
       
         
               
               
               
               
             
           
               
                   
               
             
             
               
                 Enzyme activation: 
                 95° C., 
                 10 min 
                   
               
               
                 Amplification reaction: 
                 95° C., 
                 15 s 
                 denaturation 
               
               
                   
                 60° C., 
                 30 s 
                 annealing, and elongation 
               
               
                   
                 74° C., 
                  3 s 
                 fluorescence detection 
               
               
                 40 cycles in all, 
                   
                   
                   
               
               
                 Melting curve: 
                 75~95° C. 
               
               
                   
               
             
          
         
       
     
     (3) Determination of RealTime PCR Product by Means of 1.5% Non-Denaturing (Formaldehyde Free) Agarose Gel Electrophoresis 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 microRNA RealTime PCR products 
                 2~4 μl 
               
               
                   
                 2 × Loading Buffer 
                 4 μl 
               
               
                   
                   
               
               
                   
                 The total volume was about 6~8 μl. 
               
             
          
         
       
     
     Non denaturing agarose gel: 1.2 g of agarose was added into 80 ml of 1×TBE Buffer, the mixture was heated to melt in a microwave oven and gently shaken to mix the agarose thoroughly (no suspending granules can be visually observed), 2 μl of EB (stock solution) was added when the mixture cooled to about 60° C., the solution was mixed, and then poured into a gel casting module (15×15 cm). After being placed for about 30 min at room temperature, the agarose gel would be ready to use. 
     The electrophoresis was performed at 100V for 25-30 min. 
     The results of electrophoresis shows a good specificity in microRNA RealTime PCR reaction, as shown in  FIG. 1 . 
     5. Data Collection and Processing 
     The expression values of microRNAs were converted into codes, wherein they were divided into three equal parts according to the expression levels thereof. The first one third part was given a code “1” which corresponded to the low expression level among the total expressions, the second one third part was given a code “2” which corresponded to the median expression level, and the last one third was given a code “3” corresponding to the high expression level. Then the code of each microRNA was introduced into a univariate Cox regression model to find the microRNAs associated with prognosis. Protective microRNAs for prognosis were defined as those with hazard ratio for death &lt;1. Negative-associated microRNAs for prognosis were defined as those with hazard ratio for death &gt;1[18]. After the univariate Cox proportional-hazards regression analysis was used to find microRNAs, the expression values of each microRNA were multiplied by the regression coefficients (B value) to form a linear combination used to be a prognosis risk score for each patient, wherein B value was given by the univariate Cox regression analysis. The formula was given as follows: Risk score=B1g1+B2g2+B3g3+ . . . +Bngn (B: regression coefficients, g: expression value of miRNA, n: number of miRNAs). Patients with higher risk score are expected to have poorer survival outcomes. Then patients in different groups including training group and testing group were divided into high-risk and low-risk groups using the median microRNA risk score as the cut-off point. 
     The Kaplan-Meier method was used to estimate overall survival. Differences in survival between the high-risk and the low-risk patients was analyzed. Data were normalized using U6 RNA as an internal standard. A univariate Cox regression model was used to analyze the relationship between the abundance value of each microRNA and the survival rate. A compound value was assigned to each patient according to a linear combination of the statistically significant signal value of the microRNAs derived from the univariate Cox regression analyses multiplied by the regression coefficients. The compound values were used to evaluate the prognosis of the patients. The patients were divided into two groups using the median microRNA compound value as the cut-off point, and the low-risk group had a longer survival time compared to high-risk group (P=0.005, see  FIG. 2  and  FIG. 3 ). As shown in  FIG. 2 , in the training group, the 3-year and 5-year survival of the high-risk group was 47.6% and 28.6% respectively, while the 3-year and 5-year survival of the low-risk group was 76.2%. As shown in  FIG. 3 , in the testing group, the 3-year and 5-year survival of the high-risk group was 40% and 33.6% respectively, while the 3-year and 5-year survival of the low-risk group was 74.1% and 68.8%. 
     Example 2 
     Study on the Biological Effect of MicroRNA-886-3p and MicroRNA-150 on Inhibiting the Invasion and Adhesion of Lung Cancer Cells 
     1. Experimental Procedures 
     (1) Cell Culture 
     Human small cell lung cancer cell line NCI-H446 was purchased from the Cell Center of Basic Medical Sciences, Chinese Academy of Medical Sciences, and cultured in 1640 medium containing 10% fetal bovine serum at 37° C. in a 5% CO 2  atmosphere. 
     Human non small cell lung cancer cell line NCI-H1299 was kindly provided by professor Weiguo Zhu, Department of Biochemistry and Molecular Biology, Peking University Health Science Center, and cultured in 1640 medium containing 10% fetal bovine serum at 37° C. in a 5% CO 2  atmosphere. 
     (2) miRNA Transient Transfection 
     a. Preparation of miRNA mother liquor: 250 μL 1× universal buffer was added into 5 nmol miRNA to obtain 20 μmol/L miRNA mother liquor, 
     b. The well grown cells were inoculated in a 6-well plate (without antibiotics) on the day before transfection, and the transfection was carried out when the cell density reached about 70%, 
     c. Preparation of the following complexes: Solution A: the miRNA at proper concentration was diluted into 250 μL of serum-free medium, and gently mixed. Solution B: 6 μL of Lipofectamine 2000, which had been mixed thoroughly before use, was diluted in 250 μL of serum-free medium, mixed, and incubated for 5 minutes at room temperature, 
     d. The dilution of liposome (solution B) was gently mixed with the dilution of miRNA (solution A), and incubated for 20 minutes at room temperature, 
     e. 500 μL of the mixed complexes was added into the 6-well plate. 2 mL of serum-free medium was added and then gently mixed. The original medium was removed after 6 hours, and replaced with RPMI 1640 medium containing 10% serum. 
     (3) miRNA Real-Time RT-PCR 
     a. Extraction of total RNA of samples-extraction of total RNA from cells with Trizol method 
     (I) The well grown cells were employed. When the cell density reached 80%-90%, the culture medium was poured out of the bottle, and the cells were washed twice with PBS; 
     (II) 1 ml of trizol was added into the bottle, gently shaken, and placed on ice for 15 minutes; 
     (III) The mixed solution was transferred to an Eppendorf tube pretreated with DEPC, and 200 μl of chloroform was added and mixed by shaking; 
     (IV) The solution was centrifuged at 12000 rpm for 15 min at 4° C.; 
     (V) The obtained supernatant was transferred to a new Eppendorf tube, 500 μl of isopropanol was added, gently mixed, then placed for 15 min at room temperature; 
     (VI) The solution was centrifuged at 12000 rpm for 15 min at 4° C.; 
     (VII) The supernatant was discarded, and then 1 ml of 75% ethanol was added and mixed by shaking; 
     (VIII) The solution was centrifuged at 7500 rpm for 5 min at 4° C. 
     (IX) The supernatant was discarded, and ethanol was totally evaporated in a laminar flow cabinet; 
     (X) 40˜60 μl of DEPC H 2 O was added, and the pellets were solubilized at 65° C. for 5 min; 
     (XI) The obtained sample was frozen at −20° C. for cryopreservation. 
     b. Quality assessment of total RNA 
     (I) Determination of total RNA concentration by Nanoprop (loading 2 μl of total RNA), 
     (II) Determination of RNA quality by means of 1.5% formaldehyde denaturing agarose gel electrophoresis 
     
       
         
               
               
               
             
               
               
             
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Total RNA 
                 500 ng 
               
               
                   
                 5 × Loading Buffer 
                 2 μl 
               
               
                   
                 DEPC H 2 O 
                 to 8~9 μl 
               
             
          
           
               
                   
                 The mixture was denatured at 65° C. for 5 min, 
               
               
                   
                 then put into an ice bath for 5 min. 
               
             
          
           
               
                   
                 EB(500-fold Dilution) 
                 1 μl 
               
               
                   
                   
               
               
                   
                 The total volume is about 6~8 uL. 
               
             
          
         
       
     
     Formaldehyde denaturing agarose gel: 0.45 g agarose was added to 30 ml×TBE Buffer, heated to melt in a microwave oven, gently shaken to thoroughly mix the agarose (no suspended granules can be visually observed.), then 600 ul formaldehyde was added when cooled to about 60° C. and the solution was mixed and then poured into a special gel casting module for RNA (7.5×5.5 cm). After being placed for about 30 min at room temperature, the agarose gel would be ready to use. 
     The electrophoresis was performed at 120˜430V for 15˜20 min 
     c. Reverse transcription of microRNA: 
     (I) Reaction system of reverse transcription 
     
       
         
               
               
               
             
               
               
             
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Total RNA 
                 100 ng 
               
               
                   
                 Reverse transcription  
                   1 μl(1 μM) 
               
               
                   
                 primer of microRNA 
                   
               
               
                   
                 DEPC H 2 O 
                 to 12.3 μl 
               
             
          
           
               
                   
                 The mixture was denatured at 65° C. for 5 min,  
               
               
                   
                 then put into an ice bath for 5 min. 
               
             
          
           
               
                   
                 5 × 1 st  Buffer 
                   4 μl 
               
               
                   
                 0.1M DTT 
                   2 μl 
               
               
                   
                 dNTPs 
                 0.5 μl(10 mM for each) 
               
               
                   
                 RNase Inhibitor 
                 0.2 μl(40 U/μl) 
               
               
                   
                 M-MLV 
                   1 μl(200 U/μl) 
               
               
                   
                   
               
               
                   
                 The total volume of the system was 20 μl. 
               
             
          
         
       
     
     (II) Program of reverse transcription was: 16° C. for 30 min, 37° C. for 30 min, 70° C. for 10 min, then kept at 4° C. 
     d. RealTime PCR reaction of microRNA 
     (I) Reaction system of microRNA RealTime PCR 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Template(cDNA) 
                   1 μl 
               
               
                   
                 MgCl 2    
                 1.6 μl 
               
               
                   
                 Universal sense primer of microRNA 
                 0.6 μl(10 μM) 
               
               
                   
                 Primer Specific antisense primer 
                 0.6 μl(10 μM) 
               
               
                   
                 DNA Master SYBR Green I MIX 
                   2 μl 
               
               
                   
                 Nuclease-Free H 2 O 
                 to 20 μl 
               
               
                   
                   
               
             
          
         
       
     
     (II) Program of microRNA RealTime PCR 
     
       
         
               
               
               
               
             
           
               
                   
               
             
             
               
                 Enzyme activation 
                 95° C., 
                 10 min 
                   
               
               
                 Amplification reaction 
                 95° C., 
                 15 s 
                 denaturation 
               
               
                   
                 60° C., 
                 30 s 
                 annealing, elongation 
               
               
                   
                 74° C., 
                  3 s 
                 fluorescence detection 
               
               
                 for total of 40 cycles, 
                   
                   
                   
               
               
                 Melting curve 
                 75~95° C. 
               
               
                   
               
             
          
         
       
     
     (III) Detection of RealTime PCR products by means of 1.5% non-denaturing (formaldehyde free) agarose gel electrophoresis 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 miRNA real-time PCR product 
                 2~4 μl 
               
               
                   
                 2 × Loading Buffer 
                 4 μl 
               
               
                   
                   
               
               
                   
                 The total volume is about 6~8 μl. 
               
             
          
         
       
     
     Non-denaturing agarose gel: 1.2 g of agarose was added into 80 ml of 1×TBE Buffer, the mixture was heated to melt in a microwave oven and gently shaken to thoroughly mix the agarose (no suspending granules can be visually observed), 2 μl of EB (stock solution) was added when the mixture was cooled to about 60° C., the solution was mixed, and then poured into a gel casting module (15×5 cm). After being placed for about 30 min at room temperature, the agarose gel would be ready to use. 
     The electrophoresis was performed at 100V for 25˜30 min. 
     (4) Analysis of Cell Invasion Ability 
     The principle is based on the characteristics of motility and directivity of tumor invasion. After contacting with the surface of matrix, tumor cells can move in a certain direction through a series of mechanisms. 
     a. For H446 and H1299 cells, matrigel was respectively diluted to 500 μg/mL and 1 mg/mL. 100 μl diluent was added into the upper chamber of the transwell insert of polycarbonate membrane (with 8 μm pores) and incubated for one hour at 37° C. in a 5% CO 2  incubator, and then the aqueous phase was aspirated, 
     b. The well grown tumor cells were digested and re-suspended at a certain density after 48 hours post-transfection, 
     c. 200 μl of cell suspension respectively containing 10×10 4  H446 cells or 5×10 4  H1299 cells was seeded in the upper chamber of each transwell insert, and 800 μl culture fluid containing 10% serum was added into the bottom chamber, then cells were cultured for 12 hours at 37° C. in a 5% CO 2  incubator, 
     d. The chamber was taken out and the upper layer of cells without migration were scrapped off, 
     e. Cells on the membrane were fixed with 70% methanol for 15 min, 
     f. Cells were stained with 5% crystal violet (in methanol) for 20 min, then washed with distill water, 
     g. Cells on the surface of bottom chamber were counted under a microscope, and statistically analyzed, and photographed at the same time. 
     (5) Analysis of Tumor Cell Adhesion 
     a. Fibronectin was aspirated using precooling tips under aseptic operation, and diluted to 20 μg/mL, 
     b. 50 μL diluted fibronectin was added into each well of a 96-well plate, 
     c. The 96-well plate coated with fibronectin was dried in a sterile workbench, 
     d. Cells were digested, centrifuged and resuspended with culture fluid containing 10% serum at 48 hours post-miRNA transfection, 
     e. 5×10 4  cells were seeded into each well of the 96-well plate coated with fibronectin, and 5 parallel wells were set, 
     f. After an incubation of 30, 60, 90 min or 30, 60 min respectively, H446 and H1299 cells were washed with PBS to remove non-adherent cells, and the medium was discarded in completely adhesion groups after 3 hours. Cells were fixed with 70% methanol for 10 min and dried at room temperature, then stained with 0.1% crystal violet for 20 min OD 570  was determined after decolorization with 10% SDS, which represents the adherent cells at different time points. A completely adhesion group was set up in each experimental group, 
     g. The adhesion rate was calculated with residual cells, and the cell adhesion rate=(OD value of experimental group/OD value of completely adhesion group)×100%. 
     (6) Statistical Analysis 
     The experimental data were analyzed using SPSS10.0 software package (SPSS, Chicago, Ill.) with two-sided Student&#39;s t-test, P&lt;0.05 as a significant difference. 
     2. Results 
     (1) Transfection of Lung Cell Lines with Chemically Synthetic Mature miRNAs to Overexpress Target miRNA 
     Transfection was performed using Lipofectamine 2000. For a single transfection, miR-886-3p, miR-150 or miR-AS-EGFP at a final concentration of 50 nM was used to transiently transfect H446 and H1299 cells, while for a co-transfection miR-886-3p and miR-150 at a final concentration of 37.5 nM or miR-AS-EGFP at a final concentration of 75 nM were used to transfect H446 and H1299 cells with miR-AS-EGFP as a control. At 48 hours post-transfection, cells were collected. And the expression levels of miR-886-3p and miR-150 were determined by real-time PCR. In the single transfection in H446 cell line, the expression level of miR-886-3p and miR-150 was increased by 2.5-fold and 2.9-fold respectively as compared to the control. In the co-transfection in H446 cell line, the expression level of miR-886-3p and miR-150 was increased by 1820.6-fold and 101.5-fold respectively as compared to the control. In the single transfection in H1299 cell line, the expression level of miR-886-3p and miR-150 was increased by 235.4-fold and 1723.3-fold respectively as compared to the control. In the co-transfection in H1299 cell line, the expression level of miR-886-3p and miR-150 was increased by 2736.3-fold and 2052.0-fold respectively as compared to the control. The results showed that the expression levels of miR-886-3p and miR-150 in H446 and H1299 cell lines increased significantly after the transfection, indicating that the transfection procedure and system was suitable for the corresponding research of overexpression of miRNA. 
     (2) Effect of High Expression of miR-886-3p and miR-150 on In Vitro Invasion Ability of Cells 
     In vitro invasion ability of tumor cells was studied using Transwell invasion assays. H446 cells and H1299 cells were digested and resuspened with serum free RPMI 1640 at 24 hours post-transfection, and were respectively seeded at an amount of 1×10 5  cells and 5×10 4  cells in the upper chamber of a transwell insert, while 800 μl RPMI 1640 containing 10% serum was added in the bottom chamber, then the cells were cultured for 12 hours at            7 to allow their entry into the lower layer of 8          m-pored polycarbonate membrane. Following staining with 0.5% crystal violet, cells stained purple were visible under a microscope ( FIG. 5A ,  FIG. 6A ), and cells on the lower surface of the polycarbonate membrane were counted. By calculation, the numbers of H446 cells transfected by miR-886-3p, miR-150 and miR-886-3p/miR-150 which had crossed the membrane were respectively 55.0%±8.5%, 65.7%±8.5%, 71.0%±8.5% of that of the control. The numbers of H1299 cells transfected by miR-886-3p, miR-150 and miR-886-3p/miR-150 which had crossed the membrane were respectively 73.6%±3.5%, 61.8%±11.1%, 83.7%±8.3% of that of the control. The results showed that the in vitro invasion ability of H446 and H1299 cells with high expression of miR-886-3p, miR-150 and miR-886-3p/miR-150 was obviously abated as compared to the control cells ( FIG. 5B ,  FIG. 6B ). And the difference was significant in the statistics analysis.
     (3) Effect of High Expression of miR-886-3p and miR-150 on Extracellular Matrix Adhesion of H446 and H1299 Cell Lines 
     Cell adhesion ability plays an important role in the metastasis of tumor cells. At 48 hours post-transfection, 5×10 4  H446 and H1299 cells were seeded into a fibronectin (20 μg/mL) extracellular matrix-coated 96-well plate. The cells were washed at various time points, and then the residual cells were adherent cells. The cells were fixed with 70% methanol for 10 min, stained with 0.1% crystal violet for 20 min. OD 570  value was determined after decolorization with 10% SDS. The adhesion rate was calculated, which reflected the adhesion ability to extracellular matrix. The results showed that the adhesion rate of H446 cells transfected by miR-NC, miR-886-3p, miR-150 and miR-886-3p/miR-150 respectively was 14.9%±0.9%, 11.3%±0.3%, 13.3%±0.1%, 11.4%±0.3% at 30 min; respectively was 45.1%±1.9%, 42.8%±2.2%, 45.1%±1.8%, 41.6%±1.1% at 60 min; respectively was 56.9%±1.0%, 49.0%±2.0%, 52.8%±0.5%, 47.6%±0.5% at 90 min ( FIG. 7A ). For H1299 cells transfected with miR-NC, miR-886-3p, miR-150 and miR-886-3p/miR-150, the adhesion rate respectively was 37.6%±1.5%, 34.7%±2.8%, 24.6%±3.0%, 23.4%±0.5% at 30 min; respectively were 47.1%±1.5%, 40.0%±2.1%, 36.6%±2.2%, 29.9%±4.2% at 90 min ( FIG. 7B ). As compared to the control cells, the adhesion rates of H446 and H1299 cells transfected with miR-886-3p, miR-150 and miR-886-3p/miR-150 were all reduced, and the difference was significant by a statistics analysis. The results showed that the extracellular matrix adhesion ability of H446 and H1299 cells with high expression of miR-886-3p, miR-150 and miR-886-3p/miR-150 was obviously diminished. 
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