# Identification of Unique miRNA Biomarkers in Colorectal Adenoma and Carcinoma Using Microarray: Evaluation of Their Putative Role in Disease Progression

Authors: Kothandaraman Narasimhan; Kalamegam Gauthaman; Peter Natesan Pushparaj; Govindasamy Meenakumari; Adeel Gulzar Ahmed Chaudhary; Adel Abuzenadah; Mamdooh Abdullah Gari; Mohammed Al Qahtani; Jayapal Manikandan
Journal: ISRN Cell Biology
 (2014)

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## Abstract

MicroRNAs (miRNAs) are known to be dysregulated and play a key role in cancer progression. The present study aims to identify the miRNAs associated with colorectal adenoma and carcinoma to evaluate their role in tumor progression and metastasis using microarray.In silico analysis of miRNAs was performed on five different microarray data sets that represented the genes and miRNAs expressed in colorectal adenoma and carcinoma. We identified 10 different miRNAs that were common to both colorectal adenoma and carcinoma, namely, miR9, miR96, miR135b, miR137, miR147, miR182, miR183, miR196b, miR224, and miR503. Of these, miR135b and miR147 were significantly downregulated in colorectal adenoma but upregulated  in carcinoma. In addition, we studied the gene expression  profile  associated with colorectal adenocarcinoma and identified three genes, namely, ZBED3, SLC10A3, and FOXQ1, that were significantly downregulated in colorectal adenoma compared to  carcinoma. Interestingly, of all the miRNAs and genes associated with colorectal adenocarcinoma, the myoglobin (MB) gene was identified to be under the direct influence of miR135b, showing an inverse relationship between them in adenoma and carcinoma. Most of the identified miRNAs and associated genes are involved in signaling pathways of cell proliferation, angiogenesis, and metastasis. The present study has identified putative miRNA targets and their associated gene networks which could be used as potential biomarkers of colon adenocarcinoma. Moreover, the association of miR135b and MB gene is very unique and can be considered as a lead candidate for novel cancer therapeutics.


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## Body

## 1. Introduction

Colorectal cancer is one of the most common gastrointestinal cancers that shows a rising trend due to the dietary habits and lifestyle modifications. Accumulation of genetic and epigenetic aberrations predisposes the colonic epithelium to undergo gradual transformation with loss of cellular architecture and initiation of a benign adenoma, which subsequently develops into a malignant adenocarcinoma [1]. Successful screening methods have enabled early detection and hence reduction in the mortality rate associated with colorectal adenocarcinoma [2]. In addition to the oncogenes and the tumor suppressor genes involved in cancer initiation and progression, many other small molecules such as the oncoproteins, antisense RNAs, and microRNAs (miRNAs) are also implicated in cancer and its signaling mechanisms [3].Among the small molecules involved in cancer progression and metastasis, the microRNAs (miRNAs) which are 21–25 nucleotides in length belong to the class of noncoding small RNA molecules that act as posttranslational regulators and are known to exert significant influence on most cancers [4–6]. Although the miRNAs constitute only 1–3% of the human genome, the fact that about 50% of the miRNA genes are located in the cancer associated loci indicates that these molecules are involved in either cancer progression or inhibition [5, 7]. Many different miRNAs such aslet-7, miR-24, miR-143,andmiR-192are involved in direct or indirect regulations of the KRAS or dihydrofolate signaling pathways of colorectal cancer cell proliferation [8–10]. Additionally, downregulations ofmiR-143andmiR-195 are reported to target the antiapoptotic Bcl-2 and hence induce apoptosis in colorectal cancer cell lines [11, 12]. As such the miRNAs are considered to hold great potential as diagnostic and prognostic markers of colorectal cancer [13].Currently, there are about 2019 unique mature human miRNA sequences as revealed by the miRBase 19 (miRNA database of published miRNA sequences and annotation). Given the increasing numbers of miRNAs and the fact that they have diverse expression patterns and regulations ranging from developmental biology to cancer pathology [14], it becomes a practical difficulty to validate each miRNA in order to understand their biological targets. Though high-throughput technologies such as gene chip facilitate genome-wide expression analysis of genes either in normal or diseased states, this leads to generation of enormous data which again pose great difficulty in analysis. As such bioinformatics algorithms based analysis becomes inevitable to select lead candidates and targets which could be then taken up for further validation. Recently, Li et al., 2011 [15], have used partial least square regression analysis approach for predicting miRNA targets in human colon cancer and reported that they were able to detect more miRNA-mRNA targets than a simple correlation based association in colorectal cancer. However, comparisons between numerous studies on miRNA-mRNA associations are necessary for the validation of such technology based predictions before it can be considered as an effective diagnostic/prognostic indicator.Given the scope of high-throughput technologies and the power of bioinformatics analysis, we have carried out an in-depthin silico analysis of miRNAs associated with colorectal adenoma and carcinoma. Using high-throughput gene expression as well as integrated network and pathway analyses strategies, we have critically dissected the differentially expressed miRNAs and associated gene networks in colorectal adenoma and carcinoma, respectively. We have also identified some of the predictive and therapeutic markers associated with adenoma and carcinoma and their potential to activate the adenoma-carcinoma sequel.


## 2. Methodology

Five different microarray data sets with genome-wide and miRNA expression associated with colorectal adenoma and carcinoma were used in this study. All data were obtained from recent studies on homosapiens, and the details are provided in Table1.Table 1
Overview of genome-wide transcriptomic and microRNA array data used for the study.
Study
Platform
GEO accession
Species
Source
Description
Sample size
Galamb et al., 2010 [16]
Affymetrix HGU133 Plus 2.0
GSE15960
Homo Sapiens
mRNA
Colonic epithelial cells normal, adenoma, or colorectal cancer tissues
18
Sarver et al., 2009 [17]
Illumina Human v1 MicroRNA expression beadchip
GSE18392
Homo Sapiens
miRNA
Macrodissected colon tumors and normal colon tissue
145
Arndt et al., 2009 [18]
Ambion Human_Mouse_Rat mirVANA miRNA Bioarray_1566V1
GSE10259
Homo Sapiens
miRNA
Colon cancer and normal tissues
66
Slattery et al., 2011 [19]
Agilent Human miRNA V3.0 Microarrays
NA
Homo Sapiens
miRNA
Colon and rectal tumors and normal tissue
100
Schetter et al., 2008 [20]
OSU-CCC MicroRNA Microarray Version 2.0
GSE7828
Homo Sapiens
miRNA
Colon tumor and adjacent nontumorous tissues
168
The above evidence table depicts the authors of the study, high-throughput platform used, data repository accession number, species, source material, and the number of arrays used in each study.


## 3. Genome-Wide Transcriptomic and miRNA Analysis

Raw CEL files corresponding to gene expression data GSE15960 [16] were transferred to PARTEK Genomics Suite version 6.5 0 (Partek Inc., MO, USA) and normalized using GCRMA with quantile normalization to correct for variances in distribution patterns and GC nucleotide content. Principal component analysis (PCA) was performed on all probes to visualize high dimensional data. PCA was used to demonstrate quality control as well as overall variance in gene expression between the disease states. Analysis of variance (ANOVA) was applied on the whole data set, and differentially expressed gene list was then generated using an FDR (Benjamini Hochberg) of 0.05 with 2-fold change cutoff.


## 4. Functional and Pathway Analyses

To define biological networks among the differentially regulated genes in adenoma and carcinoma, pathway analyses were performed using Ingenuity Pathways Analysis software (IPA) (Ingenuity Systems, Redwood City, CA, USA). Differentially expressed mRNAs and miRNAs that were specific for colorectal adenoma and carcinoma was imported into IPA. The association analysis between miRNAs and gene signatures from the list of imported molecules were deduced using IPA knowledgebase. Expression values were plotted against the respective miRNAs and genes to identify the expression patterns, and only those miRNAs and genes that were differentially expressed between adenoma and carcinoma were considered for further IPA analysis. The functional analysis of IPA identified the biological functions and/or diseases that were most significantly altered for the differentially expressed gene set. The canonical pathway analysis identified the pathways that were most significantly induced in adenoma and carcinoma of the colon. The significance of the association between the data set and the canonical pathways was calculated by ratio and/or Fisher’s exact test. The genes or gene products are represented as nodes, and the biological relationship between two nodes is represented by a line. Those molecules that are not linked were removed from the IPA analysis.


## 5. Results

The expression analysis of miRNAs associated with colorectal adenoma and carcinoma in the present study led to the identification of severalmiRNAs that were differentially expressed in colorectal adenoma (see Table S1A in Supplementary Material available online at http://dx.doi.org/10.1155/2014/526075) and carcinoma (Table S1B). ThesemiRNAs consisted of both tumor suppressors and oncogenes. The following oncomirs, namely,miR-21, miR-20a, miR-224,andmiR-18a, were upregulated in colorectal carcinoma, whereas the following tumor suppressors, namely,miR-29aandmiR-29c, miR-145, miR-35,andmiR-26a,were downregulated. In contrast, only one of the oncomirs, the oncogenicmiR-18a, was upregulated, and there were no known tumor suppressors that were downregulated in colorectal adenoma.Ten unique miRNAs were commonly expressed in both adenoma and carcinoma (Figure1(a)). Amongst these, bothmiR-135bandmiR-147showed differential expression patterns, being downregulated in colorectal adenoma and upregulated in carcinoma. The followingmiRNAs, namely,miR-182, miR-183, miR-196b, miR-224, miR-503,andmiR-96, were upregulated whilemiR-137andmiR-9 were downregulated in both adenoma and carcinoma, respectively. However, both upregulation and downregulation of the individual miRNAs were relatively higher in colorectal carcinoma compared to adenoma (Figure 1(b)).(a) The Venn diagram shows 10 unique miRNAs that were found to be commonly expressed in both adenoma and carcinoma of the colon. (b) Among these miRs,miR-135bandmiR-147showed differential expression patterns. Conversely, the other miRNAs are either up in adenoma (miR-182, miR-183, miR-196b, miR-224, miR-503,andmiR-96) or down in carcinoma (miR-137and miR-9).
(a)
(b)Among the genes regulated by these 10 commonly expressedmiRNAs, the zinc-finger BED domain containing 3 (ZBED3), and solute carrier family 10 (sodium/bile acid cotransporter family), member 3 (SLC10A3) genes showed differential expression patterns, being downregulated in colorectal adenoma and upregulated in carcinoma (Figure 2(a)). In addition,FOXQ1gene expression was 10-fold higher in colorectal carcinoma compared to colorectal adenoma (Figure 2(b)).Figure 2
(a) Zinc-fingerBED domain-containing 3 (ZBED3) and the solute carrier family 10 (sodium/bile acid cotransporter family) and meMBer 3 (SLC10A3) genes that were regulated by the 10 common miRNAs showed differential expression patterns between adenoma and carcinoma, respectively. (b) Similarly,FOXQ1gene expression was 10-fold higher in colon carcinoma when compared to the adenoma of the colon. (c) The upregulation of miR-224 and miR-96 in colon adenoma could cause the downregulation of bothZBED3 andSLC10A3. (d) Though both miRNAs were upregulated in colon carcinoma, the expression ofZBED3 andSLC10A3 was not influenced by these miRNAs.Comparison of 2406 genes in adenoma and 1403 genes in carcinoma revealed unique predictive markers as well as the miRNAs and their regulatory targets associated with adenoma-carcinoma sequel (Figure3(a)). Screening for the regulatory genes in both adenoma and carcinoma that are controlled by the two differentially expressed miRNAs (miR-135bandmiR-147) led to the identification ofMiR-33 being uniquely expressed in colon adenoma (Figure 3(A)). In addition, theMyoglobin (MB) gene was commonly expressed in both colorectal adenoma and carcinoma that also showed differential expression pattern being upregulated in colorectal adenoma and downregulated in carcinoma (Figure 4(a)). Furthermore, the various signalling mechanisms/interaction networks that are associated with the MB gene based on mRNA-miRNA integration analysis were identified (Figure 4(b)). These include the tumor suppressors (TP53), the angiogenic factors (VEGFA), cell proliferation, adhesion, and inflammatory molecules (EGF, VACM1, and IFNγ).(a) We compared 2406 genes in adenoma and 1403 genes in carcinoma to find the regulatory genes controlled by miRNAs that potentially mediate the carcinogenesis in adenoma-carcinoma sequel. (A) miR-33 is unique to colon adenoma stage and is responsible for the suppression of gamma-aminobutyric acid receptor beta-subunit gene (GABRB2). (B) The other miRNA which was differentially expressed was miR-147. We identified the matching partners formiR-147by comparing the same gene sets used for miR-135b. Interestingly, bothmiR-147and miR-135a/miR-135bwere not expressed in colon adenoma; however, they are both upregulated in colon carcinoma. (b) Graphical representation of adenoma-carcinoma sequel starting from normal colon and specific miRNAs and gene (oncogenes and tumor suppressors) associated with the sequel. ΩTumor suppressor, #oncogene, †oncomirs, and *mature miRNA sequence from the opposite arm of the precursor.
(a)
(b)(a)—(A) We identifiedMB gene as the differentially expressed target in colon adenoma and carcinoma that was under the control of miR-135b. (B)MB was upregulated in adenoma and downregulated in carcinoma, respectively. (C) The expression ofMB was correlated with the inverse expression pattern ofmiR-135b in adenoma and carcinoma. (b)Myoglobin interaction network in colon carcinoma. The interaction partner ofMyoglobin is represented along with the functional role they represent in colorectal cancer.
(a)
(b)The network and pathway analyses led to the identification of the cellular and molecular functions associated with miRNAs in both adenoma and carcinoma. The identified miRNAs were primarily associated with various cellular functions including cell proliferation, cell motility, cell cycle, and cell death. The miRNAs that were specific to cellular proliferation were found to be more associated with colon carcinoma (Table S2A), while those miRNAs associated with cell morphology, cell signaling, and interaction were unique to colon adenoma (Table S2B). In general, most of the miRNAs identified in the current study were associated with diseases and disorders that have been previously reported (Table S2C).The IPA network analysis identified genes such as insulin 1 (Ins1), miR-375,pyruvate dehydrogenase kinase-1(PDK1), and 3-phosphoinositide dependent protein kinase-1(PDPK1) were more represented in colon adenoma (Table S3A), whereas bruton agammaglobulinemia tyrosine kinase (BTK), CASP8 and FADD-like apoptosis regulator (CFLAR),EGF containing fibulin-like extracellular matrix protein 2 (EFEMP2), interleukin 18 (IL18), andmiR-346 were the more represented gene networks in colon carcinoma (Table S3B). As expected, the 5 gene networks identified in colon adenoma and the genes that interact with each network were primarily associated with cancer, gastrointestinal disease, carbohydrate metabolism, growth, and cellular proliferation.In addition, themiRNA-mRNAs association studies revealed distinct gene regulatory signatures in both adenoma and carcinoma. The following tumor suppressors genes, namely, phosphoinositide-3-kinase regulatory subunit 1-alpha (PIK3R1), nibrin (NBN),and neurofibromin 2 (NF2), were downregulated by miRNAs in adenomas (Table 2). Kruppel-like factor 4 (KLF4), tumor necrosis factor, alpha-induced protein 3 (TNFAIP3) and sterile alpha motif, and leucine zipper containing kinase AZK (ZAK) are some of the tumor suppressors that were downregulated in colon carcinoma. The list of miRNAs associated with these tumor suppressors in colon carcinoma is given in Table 3.Table 2
Differential expression of key miRNAs and their specific gene targets in colon adenoma.
miRNAs
miRNA expression
miRNA regulated genes
Gene expression
hsa-miR-1#Ω
Down
ABI2
Up
hsa-miR-182
Up
P
I
K
3
R
1
Ω
Down
hsa-miR-18a†Ω
Up
KRAS#
Down
hsa-miR-503
Up
P
I
K
3
R
1
Ω
Down
hsa-miR-584
Up
N
B
N
Ω
Down
hsa-miR-32
Up
N
F
2
Ω
Down
hsa-miR-96
Up
P
I
K
3
R
1
Ω
Down
ΩTumor suppressor, #oncogene, †oncomirs, and *mature miRNA sequence from the opposite arm of the precursor.Table 3
Differential expression of key miRNAs and their specific gene targets in colon carcinoma.
miRNAs
miRNA expression
miRNA regulated genes
Gene expression
hsa-let-7i*Ω
Up
AKAP5, EPHA4, KLF9, and PPP1R12B
Down
hsa-miR-107
†
Up
K
L
F
4
Ω
Down
hsa-miR-133b
Up
K
L
F
4
Ω
Down
hsa-miR-135b
Up
K
L
F
4
Ω
Down
hsa-miR-141*Ω
Up
FOXA1, NR3C1, and PRKACB
Down
hsa-miR-152
Up
K
L
F
4
Ω
Down
hsa-miR-181a*Ω
Up
AKAP5, KITLG, MARCKS, NR3C1, PLCL2, and PPP1R12B
Down
hsa-miR-29a
*Ω
Up
AKAP5,K
L
F
4
Ω
Down
hsa-miR-346
Up
K
L
F
4
Ω
Down
hsa-miR-200c
*Ω
Up
K
L
F
4
Ω, KLF9, LPAR1, MARCKS, NR3C1, PPP1R12B, and PRKACB
Down
hsa-miR-34a*Ω
Up
IL6R, KITLG
Down
hsa-miR-7
Up
K
L
F
4
Ω
Down
hsa-miR-25
Up
K
L
F
4
Ω
Down
hsa-miR-125a*Ω
Down
TNFAIP3Ω
Up
hsa-miR-137
Down
ZAKΩ
Up
hsa-miR-143*Ω
Down
ITGA6
Up
hsa-miR-145*Ω
Down
ABCC1, ATRX, ETS2, INHBB, MYC#, and SCARB1
Up
hsa-miR-195*Ω
Down
AXIN2, CXCL10, GABRE, IRAK2, KSR1, PSME3, SERPINE2, TGFBR3, UBE2S, VEGFA, ZAKΩ, and ZYX
Up
hsa-miR-26a*Ω
Down
CDK8, COL1A2, INHBB, PMAIP1, PPP1R3D, and STK4
Up
hsa-miR-34b*Ω
Down
ZAKΩ
Up
hsa-miR-9Ω
Down
DNAJB1, FOXO3#, INHBB, ITGA6, KSR1, NCOA3, NEDD4, and PPAT
Up
ΩTumour suppressor, #oncogene, †oncomirs, and *mature miRNA sequence from the opposite arm of the precursor.


## 6. Discussion

In silico based high-throughput expression analyses are being widely used to decipher the changes at the gene, mRNA, and miRNA levels, and such analyses enable us to understand their molecular interactions and networks in both normal and diseased states. Change in the miRNA expression profile is one of the common features observed in the development of cancer [3, 21]. In the present study, genome-wide transcriptomic and miRNA analysis led to the identification of ten unique mRNAs that were commonly expressed in colorectal adenoma and carcinoma. In contrast to the othermiRNAsthat were expressed and mostly upregulated in both adenoma and carcinoma, twomiRNAs, namely,miR9 andmiR137, were found to be downregulated in both colorectal adenoma and carcinoma. Decreased expression ofmiR-137which was primarily due to abnormal hypermethylation was identified to be one of the early events in colon carcinogenesis, and transfection of colon cancer cell lines withmiR-137 resulted in inhibition of cell proliferation [22]. Similarly, decreased expression ofmiR-137 was also reported with oral cancer and glioblastoma [23] indicating thatmiR-137 has tumor suppressor properties.Two of the ten miRNAs, namely,miRNA135b andmiRNA147, were differentially expressed, being downregulated in colorectal adenoma and upregulated in carcinoma. Our findings are in line with earlier studies, wheremiR-147was downregulated in colon adenoma [24] andmiR-135b was upregulated in colon carcinoma [25]. Similarly, downregulation ofmiR-135bwas found to be associated with the progression of oral carcinoma [23]. Moreover,miR-135 is also reported to target the 39 untranslated regions of adenomatous polyposis coli (APC) gene, a key multifunctional tumor suppressor in sporadic and hereditary colon carcinoma, suppresses its expression [26, 27], and induces downstream Wnt signaling [25]. Moreover,miR-135bhas been identified as a novel biomarker for pancreatic ductal adenocarcinoma by global microRNA expression profiling of microdissected tissues [28].Our study showed that upregulation ofmiR-147andmiR-135bin colon carcinoma was involved in the specific downregulation of gamma-aminobutyric acid receptor beta-subunit gene (GABRB2). MiR-33 is unique to colon adenoma stage and is responsible for the suppression ofGABRB2gene.GABRB2which is well known to be involved in transport processes of chloride channel has been previously reported as a colorectal cancer subtype classificatory [29].GABRB2was also identified as a discriminatory transcript involved in the CRC-Benign versus CRC-Crohn’s disease [29]. The miR-135b, present in the chromosome position 1q32.1 [24], could also play a role in the downregulation ofGABRB2gene.ZBED3 gene is a novel axin-binding protein that was shown to be involved in Wnt/beta-catenin signaling modulation [30]. Stage specific expression of bothZBED3 andSLC10A3 was observed in our current study and has the potential to be used as a specific biomarker to differentiate adenoma and carcinoma of the colon [31, 32]. Forkhead box Q1 (FOXQ1) transcription factor has recently been reported to play an important role in the promotion of cancer through the upregulation of several genes that promote tumor growth through angiogenesis, antiapoptotic effects [33], and epithelial-mesenchymal transition (EMT) [34]. One of the reasons for the downregulation ofZBED3 andSLC10A3 could be due to the upregulation ofmiR-224 andmiR-96 in adenoma; however, this needs experimental validation (Figure 2(a)). Probably the mechanism of upregulation of these two genes could either be a mutation or polymorphism in the mRNA coding region that could inhibit the binding of the miRNA to these genes [35]. In the presence ofmiR-33, miR-135a/miR-135bbecomes inactive. MiRNA-miRNA synergistic network is least investigated though it has been postulated to be a key factor associated with complex diseases [36]. Our results indicate a potential involvement of both positive and negative synergetic roles for miRNAs in the progression sequel associated with colon adenoma to carcinoma.We identifiedMB gene as the differentially expressed target in colon adenoma and carcinoma under the control of miR-135b. The gene was upregulated in adenoma and downregulated in carcinoma. The expression was correlated with the inverse expression pattern ofmiR-135bin adenoma and carcinoma. AlthoughMB is expressed chiefly in cardiomyocytes and oxidative skeletal muscle fibres, recent studies identified low level ofMB being expressed in various nonmuscle tissues [37]. Interestingly,MB gene has been widely implicated in epithelial cancers and given renewed importance in solid tumors [38, 39]. It was shownin vitro thatMB was expressed in hypoxic and oxidative stress conditions associated with epithelial tumors [26].MB is important in both oxygen transport and free radical scavenging, and its expression in human tumor cells promotes differentiation and inhibits metastasis [27]. Solid epithelial tumors such as colon carcinoma could take advantage of proteins such asMB to cope with hypoxic conditions and to control the metabolism of reactive oxygen and nitrogen species. Furthermore, our study showed for the first time, based on mRNA-miRNA integration analysis, the enhanced expression ofMB in adenoma and drastic downregulation in carcinoma of the colon.The downregulation of Kruppel-like family of transcription factors (KLF4) in colon cancer, and not in adenoma, was associated with the specific upregulation of known oncomirs such ashsa-miR-107as well as novel oncomirs identified in our study such ashsa-miR-133b, hsa-miR135b, hsa-miR152, hsa-miR-7,andhsa-miR-25.KLF4 functions as a tumor suppressor in several tissues, including the colon, and its specific knockdown induces epithelial-mesenchymal transition which predisposes to the development of a subset of colorectal cancers involving Wnt/beta-catenin signalling mechanisms. [40].KLF4 also directly inhibits the expression of Bmi1 in colon cancer cells [41].Phosphatidylinositol 3′-kinase p85alpha regulatory subunit 1 gene (PIK3R1) identified in the current study is also a tumor suppressor, which could potentially be downregulated through miRNA targets and requires validation. Studies on human tumor samples showed increased expression of a coordinately regulated module consisting ofPIK3R1 in advanced malignancy [42]. The PI3KR1 gene was also found to be an oncogene in human ovarian and colon tumors [43].PIK3R1 gene was also one of the genes upregulated by leptin, and this could be one of the causative factors in changing the response of colon epithelial cells possessing anAPC mutation but not normal cells. Furthermore, the genes regulating the Wnt/beta-catenin-mediated pathway includingPIK3R1 were upregulated by leptin [44], which was consistent with the progression of colon carcinogenesis.In addition,Nibrin (NBN) was found to be under the regulation of miRNAs and is associated with immortalization of colon carcinoma [45]. NBN was one of the nine genes showing altered expression in both low and high clinical stage colon carcinoma [45]. On the other hand, Neurofibromatosis 2 gene (NF2) is a candidate tumor suppressor gene in chromosome 22.q locus [46], and the colon cancers commonly have allelic losses of chromosome 22q. Consequently, NF2 gene was found to be the target of potential miRNAs in colon carcinoma in our study.


## 7. Conclusions and Future Directions

In the present study, we have exploited the high-throughput expression analyses strategies to critically delineate the mRNA as well as miRNA expression profiles of adenoma and carcinoma of the colon. Besides, we have identified novel miRNA regulatory networks that regulate the transcription of mRNAs required for the adenoma and carcinoma sequel. It was significant to note that none of the miRNAs and their gene targets identified either in adenoma or carcinoma had overlapped in their expression patterns. This shows that specific miRNAs are expressed in a stage specific manner and regulate their candidate genes in colon adenoma and carcinoma, respectively. These transcriptional networks that regulate genes involved in the molecular functions associated with the differentiation of adenoma into carcinoma provide the predictive markers of colon adenoma-carcinoma sequel and together with RNAa/RNAi strategies to increase the expression of the tumor suppressor genes as well as silencing oncogenes would add tremendously to early detection and management of colon carcinoma.



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*Source: 526075-2014-04-22.xml*