| Colorectal cancer is one of the most common malignant tumors in digestive tract.The incidence of colorectal cancer is increasing year by year in China,and there is a trend of younger age.Studying its pathogenesis is helpful to search for new screening and treatment strategies.In recent years,miRNAs have been shown to act as oncogenes or tumor suppressor genes in a variety of malignant tumors,and more and more miRNAs have been identified as tumor suppressors in the development of colorectal cancer.Recent studies have shown that miR-519b-3p plays an important role in the progression of many malignant tumors,but the mechanism and pathway of miR-519b-3p in colorectal cancer are not clear.WNT signaling pathway is one of the most important pathways in colorectal cancer research,which is a classical pathway regulating cell proliferation and growth.Objective: To investigate the expression of miR-519b-3p in human colorectal cancer tissues and cells,and to explore the biological function and mechanism of miR-519b-3p in colorectal cancer.Methods: 1.The expression of miR-519b-3p in colorectal cancer tissues,its adjacent tissues,colorectal carcinoma epithelial cells and normal colon epithelial cells were detected by real-time quantitative PCR(q RT-PCR).2.After constructing miR-519b-3p high expression group and knock-down group,the potential effects of miR-519b-3p on the proliferation and invasion of cancer cells were detected by CCK-8 and transwell experiments.3.The expression levels of downstream proteins of Wnt pathway were detected by Elisa and Western Blot to investigate the effect of miR-519b-3p on WNT pathway.4.The miR-519B-3P target gene was predicted using bioinformatics methods,and the double luciferase reporter gene test demonstrated that miR-519b-3p targets with u Mt CK m RNA.5.We detected the expression of UMt CK in colorectal cancer and normal adjacent tissues by q RT-PCR.6.Western Blot and q RT-PCR were used to examine the relationship between the expression of u Mt CK and miR-519b-3p in colorectal cancer cells.7.After transfection,CCK-8 and transwell were used to detect whether the upregulation of u Mt CK could reverse the effect of miR-519b-3p on the biological function of cancer cells.8.Topflash reporter gene was used to detect whether u Mt CK could modulate the effect of miR-519b-3p on WNT pathway activity in cancer cells.Results: 1.The results of q RT-PCR showed that miR-519b-3p was low expressed in the colorectal cancer tissues and cells,and was significantly different from that in the adjacent normal tissues and normal colon cells.2.The proliferation and invasion of cancer cells in the high-expression group were the weakest,which was significantly different from that in the knock-down group and the negative control(NC)group.3.In miR-519b-3p overexpression group,knock-down group and NC group,the expression of WNT pathway and downstream genes cmyc and Cyclin D1 decreased significantly with high miR-519b-3p expression.4.Double luciferase reporter gene test suggested that miR-519b-3p significantly inhibited the luciferase activity of wild-type u Mt CK reporter gene,but not the mutant type.5.q RT-PCR showed that u Mt CK was over-expressed in colorectal cancer tissue,which was significantly different from normal adjacent tissue.6.In miR-519b-3p overexpression group,knock-down group and NC group,expression of u Mt CK was negatively correlated with miR-519b-3p expression,and there were significant differences between groups.7.After constructing miR-519b-3p mimics + u Mt CK group,miR-519b-3p mimics + vector group and NC group,CCK-8 and transwell test were performed,the results showed that the ability of cell proliferation and invasion in NC group was the strongest,followed by miR-519b-3p mimics + u Mt CK group,and that in miR-519b-3p mimics + vector was the weakest in Rko and DLD-1 cells.There was significant difference in the ability of proliferation and invasion between u Mt CK group and vector group.8.In the miR-519b-3p mimics + u Mt CK group,miR-519b-3p mimics + vector group and NC group,WNT pathway activity was detected by the TOPFlash reporter gene test.WNT pathway activity was highest in NC group and lowest in mimics+ vector group,and slightly lower in mimics + u Mt CK group,the difference between the two groups was significant.Conclusion: Low expression of miR-519b-3p in colorectal cancer and high expression of miR-519b-3p can inhibit the proliferation and invasion of colorectal cancer cells,this biological effect is mediated by miR-519b-3p targeting u Mt CK m RNA to regulate the Wnt signal pathway. |
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