miR-340靶向Lgr5基因抑制结肠癌细胞增殖及β-catenin通路的研究

MiR-340 Inhibits Proliferation andβ-Catenin Pathway of Colon Cancer Cell via Targeting Lgr5 Gene

目的研究miR-340靶向富含亮氨酸重复序列的G蛋白偶联受体5(Lgr5)基因抑制结肠癌细胞增殖及β-连环蛋白(β-catenin)通路的作用。方法培养人结肠癌细胞株HT29、SW480、LoVo及正常肠上皮细胞HIEC,检测miR-340的表达水平;将SW480细胞分为miR-NC组(转染miR-NC mimic)、miR-340组(转染miR-340 mimic)、pcDNA3.1组(转染pcDNA3.1质粒),pcDNA3.1+miR-340组(转染pcDNA3.1质粒及miR-340 mimic)、pcDNA3.1-Lgr5+miR-340组(转染pcDNA3.1-Lgr5质粒及miR-340 mimic),MTS法检测增殖活力A490,荧光定量PCR检测miR-340的表达水平,Western blot检测Lgr5、β-catenin、cyclinD1的表达水平,双荧光素酶报告基因验证miR-340靶向Lgr5。结果HT29、SW480、LoVo细胞中miR-340的表达水平均低于HIEC细胞,且SW480细胞中miR-340的表达水平最低;miR-340组的A490水平、Lgr5、β-catenin、cyclinD1的表达水平、Lgr5野生型双荧光素酶报告基因的荧光活力均低于miR-NC组;pcDNA3.1+miR-340组的A490水平、Lgr5、β-catenin、cyclinD1的表达水平均低于pcDNA3.1组,pcDNA3.1-Lgr5+miR-340组的A490水平、Lgr5、β-catenin、cyclinD1的表达水平均高于pcDNA3.1+miR-340组。结论miR-340能够抑制结肠癌细胞的增殖,靶向Lgr5基因并抑制β-catenin通路是可能的分子机制。

Objective To study the inhibitory effect of miR-340 on proliferation andβ-catenin pathway of colon cancer cell via targeting leucine-rich repeat containing G protein-coupled receptor 5(Lgr5)gene.Methods Human colon cancer cell lines HT29,SW480,LoVo and normal intestinal epithelial cell HIEC were cultured.The expression level of miR-340 was detected in these cells.SW480 cells were divided into the miR-NC group(transfected with miR-NC mimic),miR-340 group(transfected with miR-340 mimic),pcDNA3.1 group(transfected with pcDNA3.1 plasmid),pcDNA3.1+miR-340 group(transfected with pcDNA3.1 plasmid and miR-340 mimic),and pcDNA3.1-lgr5+miR-340 group(transfected with pcDNA3.1-lgr5 plasmid and miR-340 mimic).Proliferation viability A490 was detected by MTS assay,the expression level of miR-340 was detected by PCR,the expression levels of Lgr5,β-catenin and cyclinD1 were detected by western blot,and miR-340 targeting Lgr5 was verified by a double luciferase reporter gene assay.Results The expression levels of miR-340 in HT29,SW480 and LoVo cells were lower than that in HIEC cells,and the expression level of miR-340 in SW480 cells was the lowest.The level of A490,the expression levels of Lgr5,β-catenin,cyclinD1 and fluorescence activity of Lgr5 wild-type double luciferase reporter gene in the miR-340 group were lower than those in the miR-NC group.The level of A490,the expression levels of Lgr5,β-catenin,and CyclinD1 in the pcDNA3.1+miR-340 group were lower than those in the pcDNA3.1 group.The level of A490,the expression levels of Lgr5,β-catenin,and CyclinD1 in the pcDNA3.1-Lgr5+miR-340 group were higher than those in the pcDNA3.1+miR-340 group.Conclusion miR-340 can inhibit the proliferation of colon cancer cells,in which targeting Lgr5 gene and inhibitingβ-catenin pathway could serve as a possible molecular mechanism.