miR-377靶向ZEB2调节结肠癌细胞迁移、侵袭和上皮-间质转化

miR-377 targets ZEB2 to regulate migration,invasion and epithelial-mesenchymal transition of colon cancer cells

目的:探讨miR-377对结肠癌细胞迁移、侵袭的影响及潜在作用机制。方法:RT-qPCR检测结肠癌细胞系HT29、SW480、SW620和HCT116和人小肠上皮细胞HIEC miR-377表达。将miR-377 mimic、阴性对照物、pmirGLO-wt-ZEB2、pmirGLO-mut-ZEB2转染至HCT116细胞,双荧光素酶报告实验检测miR-377与ZEB2的靶向作用,伤口愈合实验、Transwell、Western blot检测miR-377和ZEB2对细胞迁移、侵袭、上皮-间质转化相关蛋白表达的影响。结果:与HIEC细胞相比,结肠癌细胞系miR-377表达降低(P<0.05)。与对照组相比,miR-377 mimic和pmirGLO-WT-ZEB2共转染的HCT116细胞荧光素酶活性显著降低(P<0.05),但ZEB2结合位点突变逆转了miR-377 mimic对荧光素酶活性的抑制作用(P>0.05);miR-377 mimic显著降低了HCT116细胞ZEB2蛋白表达(P<0.05)。与NC+Vector组相比,miR-377+Vector组细胞划痕愈合率、侵袭细胞数显著降低(P<0.05),E-cadherin蛋白表达显著升高(P>0.05),N-cadherin、Vimentin蛋白表达显著降低(P<0.05)。与miR-377+Vector组相比,miR-377+ZEB2组细胞划痕愈合率、侵袭细胞数显著高于miR-377+Vector组(P<0.05),E-cadherin蛋白表达显著降低(P<0.05),N-cadherin、Vimentin蛋白表达显著升高(P<0.05),miR-377+ZEB2组与NC+Vector组上述指标差异无统计学意义(P>0.05)。结论:miR-377通过靶向作用于ZEB2调节结肠癌细胞上皮-间质转化相关蛋白表达,进而影响结肠癌细胞迁移、侵袭,为结肠癌临床治疗提供了新的策略。

Objective:To investigate effect of miR-377 on migration and invasion of colon cancer cells and its potential mechanism.Methods:RT-qPCR was used to detect expression of miR-377 in colon cancer cell lines HT29,SW480,SW620 and HCT116and human small intestinal epithelial cells HIEC.HCT116 cells were transfected with miR-377 mimic,negative control,pmirGLO-wtZEB2 and pmirGLO-mut-ZEB2.Luciferase reporter assay was used to detect targeting effect of miR-377 and ZEB2.Effects of miR-377and ZEB2 on cell migration,invasion and epithelial-mesenchymal transition related proteins were detected by wound healing test,Transwell and Western blot.Results:Compared with HIEC cells,expression of miR-377 in colon cancer cell lines was decreased(P<0.05).Luciferase activity of HCT116 cells co transfected with miR-377 mimic and pmirGLO-wt-ZEB2 was significantly lower than that in control group(P<0.05),but mutation of ZEB2 binding site reversed inhibitory effect of miR-377 mimic on luciferase activity(P>0.05).miR-377 mimic significantly decreased expression of ZEB2 protein in HCT116 cells(P<0.05).Compared with NC+Vector group,wound healing rate and number of invasive cells were decreased in miR-377+Vector group(P<0.05),expression of E-cadherin protein was increased(P<0.05),protein expressions of N-cadherin and Vimentin were significantly decreased(P<0.05).Compared with miR-377+Vector group,wound healing rate and number of invasive cells were increased in miR-377+ZEB2 group(P<0.05),expression of E-cadherin protein was decreased(P<0.05),protein expressions of N-cadherin and Vimentin were significantly increased(P<0.05).There was no significant difference of above indexes between miR-377+ZEB2 group and NC+Vector group(P>0.05).Conclusion:miR-377 regulates expressions of epithelial-mesenchymal transition related proteins in colon cancer cells by targeting ZEB2,and affects migration and invasion of colon cancer cells,which provides a new therapeutic strategy for clinical treatment of colon cancer.