Screening And Validation Of The Target Genes Of MicroRNAs And The Molecular Biology Mechanism Of MiR-424during Colorectal Carcinogenesis

IntroductionColorectal adenocarcinoma (CRC) is one of the most common malignant tumors and a major cause of cancer mortality in digestive system. The etiology of colorectal cancer is multifactorial, genetic and long-term formation of the complex lesions in the process. Early discovery, Early diagnosis, early and standard operation treat is the key to improve survival rates for colorectal cancer. Therefore, further investigation into the molecular pathogenesis during colorectal cancer early pathological stage is particularly important. Recently, microRNA (miRNA) has received the greatest attention due to its important role in the regulation of gene expression.MicroRNA (miRNA),18-to25-nucleotide, endogenous non-coding single-stranded RNA molecule, carries out its biological functions by binding to the3’ untranslated regions (UTRs) of its target messenger RNAs (mRNAs) and thereby repressing their expression. The fact that miRNAs widely expressed in various species and tissues indicates that miRNAs play fundamental roles in many biological processes, including development and cell differentiation,progression, apoptosis and cancer development,and progression. Some studies confirmed that microRNAs can act as oncogenes or tumor suppressors, and directly effect on human tumors (such as lung cancer, breast cancer, brain cancer, liver cancer, colorectal cancer, lymphoma, etc.), they are important molecules in tumor occurrence and development process. Although aberrant expression of miRNAs has been shown to associate with the colorectal cancer initiation and transformation by regulate their target mRNAs. Most of microRNAs and their role in the target genes and specific biological function remains to be further research.In our previous studies,we had combined laser capture microdissection (LCM) with genome-wide miRNA analysis, and discovered40miRNAs signatures at the multisteps of colorectal carcinogenesis by used225frozen surgical specimens.In this study, we first screened and validated the potential target genes of miRNAs in the transformation and progression of colorectal cancer, and then chose one of the miRNAs,miR-424,for further study of its expression in clinical samples of colon lesions using in situ hybridization. We then validated the candidate target genes of miR-424and explored biological behavior of miR-424in colorectal cancer cell line, revealed the biological function of miR-424in colorectal carcinogenesis, and provided the molecular theory basis for the early emergence of colorectal cancer. Moreover, the functional study of miR-424may shed the light on the new target for CRC diagnosis and treatment.Part I Screening the potential target genes of miRNAs in the transformation and progression of colorectal cancerObjective:To predict and screen the target genes for candidate microRNA biomarkers in the transformation and progression of colorectal cancer.Methods:The target genes for each of40microRNA biomarkers were predicted through the gateway miRecords. The genes that were predicted by at least4of11databases were selected as targets. The KEGG database was used to map the predicted targets of microRNAs to colorectal cancer-associated pathways. In59frozen colorectum tissues (n=18normal tissues, n=19adenomas and n=22Dukes’A/B stage adenocarcinoma), quantitative RT-PCR was performed on the potential target genes and their corresponding miRNAs. The corrected p value<0.05in the unpaired t-test and fold change≥2.0were used to determine the statistical significance.Results:Putative targets for microRNA biomarkers were identified through miRecords and KEGG databases.There were58target genes of27microRNAs in colorectal cancer-associated signaling pathways, including Wnt, TGF-beta, MAPK and p53signaling pathway. On the tissue level,20significant differential expressed target genes were identified in different tissues. There was negative correlation between thirteen of them and their corresponding eight miRNAs (miR-135b, miR-182, miR-183, miR-34a, miR-424, miR-552, miR-769-5p and miR-96). Through the further verification by qRT-PCR, we found that there was negative correlation between five of the miRNAs (miR-135b, miR-182, miR-183, miR-424and miR-96) and their nine candidate target mRNAs (NODAL, SMAD4, MET, CYCS, PDGFRA, MAP2K1, SMAD3, MAP2K4and WNT7A). Additionally, we found that miR-424could differentiate adenomas from the early colorectal carcinomas(p<0.001) better than other four miRNAs.Conclusions:mRNAs expression were showed significant differences in different tissues of colorectum, some of the mRNA expression showed negative correlation with their corresponding miRNAs expression. The combination of miRecords and KEGG database is a good method to predict and select signaling-related target genes for microRNA. The target genes with the negative correlation of their microRNA expression shall be selected for further functional studies.Part ⅡA differential analysis of miR-424expression in early colorectal cancer and precancerous lesions by in situ hybridization.Objective:To Detect expression of miR-424in the normal intestinal epithelium, adenomas (low-grade intraepithelial neoplasia and high-grade intraepithelial neoplasia) and early colorectal cancer by in situ hybridization.Methods:We detected the expression of miR-424by in situ hybridization in30FFPE colorectal tissue specimens, including7samples of low-grade intraepithelial neoplasia(LGIN),8samples of high-grade intraepithelial neoplasia(HGIN),15samples of and adjacent normal tissue for control study. Combined with the spectrum detection technology of CRi-Nuance multispectral microscope imaging system and inForm software, quantitative analysis was performed on miR-424positive expression scope(cytoplasm pixel area) and cytoplasm optical density in the tissue of colorectal cases.Results:The miR-424average expression area in the cytoplasm of adenomas and early colorectal cancer,was larger than their adjacent normal tissue. The expression range of miR-424significantly increasing in the cytoplasm from Dukes’A/B stage adenocarcinomas to their adjacent nprmal tissue (A=1.16), HGINs to their adjacent normal tissue (Δ=1.56), LGINs to their adjacent normal tissue (Δ=1.56),and Dukes’ A/B stage adenocarcinomas adjacent normal tissue to HGINs adjacent normal tissue (Δ=1.32)(p<0.05); The cytoplasm optical density of miR-424increased from Dukes’ A/B stage adenocarcinomas to their adjacent normal tissue (Δ=1.63) and LGINs (Δ=1.34),and HGINs to LGINs(A=1.29)(p<0.05) Conclusions:The application of multispectral microscope imaging system in the quantitation of microRNA signal validly distinguished the different expression level of miR-424in the tested samples and showed statistic significance in some of the interested criteria (between early colorectal cancer and its adjacent normal tissue, early colorectal cancer and LGIN, HGIN and LGIN, HGIN and adjacent normal tissue of early colorectal cancer), except no statistic significance was found between early colorectal cancer and HGINs tissue samples.PartⅢ Identification of candidate target genes and biological functions of miR-424Objective:To Validate relationship between miR-424and their candidate target genes at cellular level, and investigate the biological functions of miR-424in colon cancer cell DLD1.Methods:miR-424highly expression cancer cell model, DLDl-LV3-pre-miR-424were established by transfected the lentivirus into human colon cell line DLD1, and also set up negative control. The level of mRNAs was determined by qRT-PCR, luciferase activity was validated by Luciferase Reporter Assay and the expression level of proteins by Western blot. Cell growth was evaluated by CCK-8proliferation assay. Cell invasion and migration potential were evaluated by transwell invasion and migration assay.Results:The luciferase reporter assay revealed that the luciferase activity with MAP2K1AD3and WNT7A3’UTR were significantly inhibited by miR-424. We did not find any effects of LV3-pre-miR-424on MAP2K1,SMAD3and WNT7A mRNA level, while SMAD3and WNT7A protein level descreased. The CCK-8proliferation assay showed that cell growth ability was no significant change in DLD1-LV3-pre-miR-424compared with negative control cell. The transwell invasion and migration assay showed that overexpression of miR-424in colon cancer cell resulted in significant promotion of cell invasion and migration compared with negative control cell. Conclusions:miR-424directly inhibited expression of SMAD3and WNT7A,and suppressed their protein expression levels. Overexpression of miR-424promoted colon cancer cell invasion and migration.