MiR-340Regulates Oxaliplatin-induced Epithelial-mesenchymal Transition By Targeting Bmi-1in Colorectal Cancer Cells

ObjectiveTo explore the impact of miR-340expression target Bmi-1to the cell model of epithelial-mesenchymal transition (EMT), which was induced by oxaliplatin in colorectal cancer cells, in order to clarify the mechanisms of epithelial-mesenchymal transition in colorectal cancer and provide new theoretical basis for intervention in colorectal cancer metastasis.Methods1. The colorectal cancer cell line HT29was induced by different concentrations of oxaliplatin, by observing the changes in cell morphology, the expression of epithelial-mesenchymal marker and the change of cell proliferation, migration and invasion ability to verify whether the cell occurred EMT.2.Transient transfections of miR-340mimics or miR-negative control mimics were carried out in oxaliplatin-resistant colorectal cancer HT-29cells(HT29-OxR)using Lipofectamine2000following the manufacturer’s instructions. miR-340expression was further evaluated by RT-qPCR;Using Transwell experiments to observe the migration and invasion ability of the cells after transfection; Cells proliferation were detected by MTT; The biomarkers of EMT, E-cadherin and vimentin were measured by RT-qPCR, at the same time, Western blot was used to detect E-cadherin and vimentin from the level of protein expression.3. Bioinformatics analysis by TargetScan and miRanda indicated that Bmi-1is a potential target of miR-340. Luciferase reporter assay was carried out using the vectors containing wild type (WT)-Bmi-l3’-UTR sequences or mutant (MUT)-Bmi-13’-UTR sequences. Cells were transiently co-transfected with miR-340mimics/miR-negative control mimics and WT-Bmi-13’-UTR vector/MUT-Bmi-13’-UTR vector. Luciferase activities were measured with the Dual-Luciferase assay kit according to manufacturer’s instructions24h after transfection. RT-qPCR and Western blot were performed after transfections with miR-340mimics or miR-negative control mimics to detect the Bmi-1expression in both mRNA and protein levels.Resultl.The cell morphology of colorectal cancer cell lines HT29were significantly different compared to normal cells.HT29cells were grown and gathered in clusters and tight adhesion between cells,however,the cells of HT29-OxR were spindle-shaped,loss of adhesion between cells and a large number of pseudopods formed. These changes were the character of mesenchymal cells. Real-time quantitative PCR (RT-qPCR) showed that in HT29-OxR cell lines,epithelial marker E-cadherin was decreased,while the mesothelial marker vimentin was increased. In addition,Transwell experiments showed,the migration and invasion ability of HT29-OxR was increased compared with the control group. The results of wound healing assays were consistent with the Transwell assays’results. The above results showed that the HT29-OxR cells have occurred EMT and the migration and invasion ability the cells were increased.2. The expression of miR-340in HT29-OxR cells that transfected with miR-340mimics were significantly higher than the group which were transfected with miR-negative control mimics (P＜0.01);HT29-OxR cells were transfected with miR-340mimics, using qTR-PCR measured epithelial marker E-cadherin was increased, while the mesothelial marker vimentin was reduced, which exhibited characteristics of epithelial cells; MTT assay showed miR-340can promote the proliferation of HT29-OxR cells. EMT was reversed.3. Bioinformatics analysis by TargetScan and miRanda indicate that3’-UTR of Bmi-1 contains predicted binding site for miR-340. Luciferase reporter assay showed that co-transfection of miR-340mimics significantly suppressed the luciferase activity of the reporter containing wild-type3’-UTR but did not suppress the mutant reporter(P <0.01).These data reveal that Bmi-1is a direct functional target of miR-340. Consistent with these results,transfection of miR-340mimics diminished the expression of Bmi-1in both mRNA and protein levels of HT29-OxR cells.Conclusion1. Different concentrations of oxaliplation can induce the colorectal cancer cell line HT29occurred EMT.2. The high expression of miR-340can inhibit the migration and invasion ability of the cells in the modern of EMT,and the EMT was reversed.Bmi-1can be used as target gene of miR-340and affect the transformation of EMT. This is of great significance in searching important molecular targets which can reverse the EMT of colorectal cancer,and intervene the colorectal cancer metastasis at the key step.