Effects And Potential Mechanisms Of MicroRNA-194on Epithelial Mesenchymal Transition Of Colorectal Cancer

Background:Colorectal cancer is the most common gastrointestinal malignancy, which seriously threat to human health. Currently, multidisciplinary treatments based on surgical resection is rapid development in colorectal cancer. Especially chemotherapy, molecular targeted drugs are familiar in clinical application, which effectively improve the overall survival for the colorectal cancer patients. However, statistics showed that the incidence and disease-related mortality rate is still rising obviously. Diastant metastases is a key factor affecting prognosis and death for colorectal cancer patients.The formation of metastases is a multistep process, in which malignant cells disseminate from the primary tumour to colonize distant organs. This is a highly inefficient and complex process. It is now recognized that the epithelial mesenchymal transition (EMT) may represent a critical component permitting the progression of carcinomas towards invasive and metastatic disease. Colorectal cancer carcinogenesis has been extensively studied at a molecular level in recent years and has recently entered the era of microRNAs (miRNAs). MiRNAs are a family of small, highly conserved non-coding RNAs that post-transcriptionally regulate gene expression, which act as endogenous suppressors of gene expression through imperfect binding of RNA-induced silencing complex (RISC) to the target mRNAs. MiRNAs have been studied in colorectal cancer as potential biomarkers of diagnosis, prognosis or response to treatment as well as colon cancer cell proliferation, apoptosis and angiogenesis. However, researches on miRNAs mediated EMT are still rare. Our studay group previously concerned reaearch on the relationship between miRNAs and chemotherapy-resistance, which showed up-regulation of miR-200c with transfection of miR-200c mimics in breast cancer cells could enhance the chemosensitivity to epirubicin and reduce expression of multidrug resistance-1mRNA and P-glycoprotein. In this study, our group will focus on the effects and potential mechanisms of microRNA-194on EMT process of colorectal cancer.Methods and resultsFirstly, our research group collected40specimens of colorectal cancer, using stem-loop RT-qPCR technique to evaluate the relative expression of miR-194in different specimens and add up the correlation with clinicopathological features. The results showed that miR-194expression was significantly increased in lymph node-positive colorectal cancers. However, miR-194expression level was not significant correlation to gender, age, tumor location, tumor size, degree of differentiation and T stage. The results suggest that miR-194may play an important role in metastasis of colorectal cancer.Secondly, our team looking for appropriate cell model to the further study on the effects and potential mechanisms of microRNA-194in colorectal cancer invasion and metastasis. We found miR-194was significantly higher expression in SW620than SW480cell line. Subsequent wound healing and Transwell assay confirmed the ability of migration and invasion of SW620cells was significantly increased than SW480cells. Further studies showed that the expression of MMP-2not only in mRNA but protein level was significantly higher in SW620v.s SW480, however, MMP-9mRNA expression was no significant difference. The expression of epithelial cell marker E-cadherin was significantly higher in SW480, mesenchymal cell marker Vimentin was contrary. Therefore it confirmed that MMP-2, E-cadherin, Vimentin play an important role in colorectal cancer EMT process.Next, miR-194plasmid vector is constructed and stably transfected into the relatively low expression cell lin SW480. We successfully established a stably transfected cell line and named miR-194-480by screening high expression of miR-194cell clones. Morphological observation showed the shape of miR-194-480was significantly into fusiform compared with the non-transfected cell line SW480, which illustrate the visible interstitial changes. MTT assay showed overexpression of miR-194had no significant effect in cell proliferation, while, wound healing and Transwell assay confirmed the ability of migration and invasion of miR-194-480was significantly increased. Therefore, we believe that miR-194may have little effect in proliferation of colon cancer cells, but manifest increase the ability of migration and invasion. RT-qPCR, Western Blot and gelatin zymography assay demonstrated overexpression of miR-194can increase MMP-2protein expression and activity involved in degradation of the extracellular matrix, while decreased expression of E-cadherin, however Vimentin expression was significantly enhanced. Therefore, we confirmed that overexpression of miR-194play an important role in colorectal cancer EMT process by changing the expression of MMP-2, E-cadherin and Vimentin.Cytoskeletal staining experiment confirmed that overexpression miR-194can increase the number of cell microfilaments. The result showed that miR-194can affect cell microfilament, changing cytoskeleton, and ultimately increase the ability of migration and invasion in colon cancer cell.Our group next concern the cellular signal transduction pathways. By predicting of bioinformatics screening as TargetScan, miRanda, RNA22software, PKC may be a target of miR-194. More and more research concerned PKC activated the ERK pathway. We predict whether miR-194can increase PKC expression, thereby activating the ERK pathway, finally involved in colorectal cancer EMT transformation. But we got a negative result. Analyze the reasons may be involved in other pathways and miR-194feedback inhibition of PKC-alpha, of course, can not be ruled out experimental techniques are not mature enough. Analysis of the reasons, miR-194may be involved in other pathways then feedback inhibition of PKC-alpha, and of course can not rule out our immature experimental techniques. However, we are unable to further delve into the subject because of time constraints.Conclusions:MiR-194expression was significantly increased in lymph node-positive colorectal cancers, however, which was not significant correlation to gender, age, tumor location, tumor size, degree of differentiation and T stage. MiR-194play an important role in colorectal cancer EMT process by changing the expression of MMP-2, E-cadherin and Vimentin. Overexpression miR-194can increase the number of cell microfilaments. PKC-alpha pathway may not be the core signaling pathway in miR-194-mediated EMT process in colorectal cancer.