miR-1180基因沉默介导Wnt通路增强人结肠癌耐药细胞株THC8307/L-OHP化疗的敏感性  

MiR-1180 gene silencing mediated Wnt pathway enhances the chemosensitivity of human colon cancer drug-resistant cell line THC8307/L-OHP

作　　者：苍宏宇 郝灵芳[2] 梁润 CANG Hong-yu;HAO Ling-fang;LIANG Run(Department of Oncology,Inner Mongolia Autonomous Region People′s Hospital,010010;Department of Oncology,Huhhot First Hospital,010030)

机构地区：[1]内蒙古自治区人民医院肿瘤内科,010010 [2]呼和浩特市第一医院肿瘤内科,010030

出　　处：《现代消化及介入诊疗》2022年第4期463-468,共6页Modern Interventional Diagnosis and Treatment in Gastroenterology

基　　金：呼和浩特市科技计划项目(2018-社-7-4)。

摘　　要：目的探讨微小核糖核酸-1180(miR-1180)基因沉默介导Wnt通路增强人结肠癌耐药细胞株THC8307/奥沙利铂(L-OHP)化疗敏感性的作用。方法设计并构建miR-1180 inhibitor、NC inhibitor慢病毒载体,采用脂质体转染法将其转染至人结肠癌耐药细胞株THC8307/L-OHP内,分别记为沉默组和对照组,另取未转染细胞记为空白组,每组6个复孔。转染48 h后,荧光显微镜下观察计算沉默组和对照组细胞转染效率。分别在细胞培养液中加入0、3.13、6.25、12.5、25、50μmol/mL的L-OHP作用48 h,噻唑蓝(MTT)法检测各组细胞活力和对L-OHP的半数抑制浓度(IC_(50));流式细胞术检测各组细胞凋亡率;实时定量聚合酶链反应(RT-qPCR)检测各组细胞miR-1180表达及Wnt、β-连环蛋白(β-catenin)、细胞周期蛋白D1(CyclinD1)、多药耐药基因1(MDR1)、抗凋亡因子(Bcl-2)信使核糖核酸(mRNA)表达;蛋白质免疫印迹法(WB)检测各组细胞Wnt、β-catenin、CyclinD1、MDR1、Bcl-2蛋白表达;双荧光素酶活性实验验证miR-1180靶向调控Wnt。结果对照组和沉默组转染效率分别为(93.51±1.89)%、(94.09±2.26)%。与空白组和对照组比,沉默组细胞活力、IC_(50)值降低,细胞凋亡率升高,差异均有统计学意义(P<0.05),且空白组和对照组细胞活力、IC_(50)值、细胞凋亡率差异均无统计学意义(P>0.05);与空白组和对照组比,沉默组miR-1180表达、Wnt、β-catenin、CyclinD1、MDR1、Bcl-2mRNA及蛋白表达均降低,差异均有统计学意义(P<0.05),且空白组和对照组miR-1180表达、Wnt、β-catenin、CyclinD1、MDR1、Bcl-2mRNA及蛋白表达差异无统计学意义(P>0.05)。结论miR-1180基因沉默可显著降低人结肠癌耐药细胞株THC8307/L-OHP的细胞活力,提高化疗敏感性,推测是通过靶向Wnt信号通路,抑制Wnt、β-catenin、CyclinD1、MDR1、Bcl-2的表达实现此作用的。Objective To investigate the role of Wnt pathway mediated by microRNA-1180(miR-1180)gene silencing in enhancing the chemosensitivity of human colon cancer drug-resistant cell line THC8307/oxaliplatin(L-OHP).Methods miR-1180 inhibitor and NC inhibitor lentivirus vectors were designed and constructed.They were transfected into human colon cancer drug-resistant cell line THC8307/L-OHP by liposome transfection method.They were recorded as silencing group and control group respectively,and the non-transfected cells were recorded as blank group,with 6 wells in each group.The transfection efficiency of cells in silencing group and control group was observed and calculated under fluorescence microscope 48 hours after transfection.L-OHP concentrations of 0,3.13,6.25,12.5,25 and 50μmol/ml were added to the cell culture medium respectively for 48h,the cell viability and the half inhibitory concentration(IC_(50))of L-OHP in each group were detected by methyl thiazolyl tetrazolium(MTT).The apoptosis rate was detected by flow cytometry.Real time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of miR-1180 and Wnt,β-catenin,Cyclin D1,multidrug resistance gene 1(MDR1),Bcl-2 mRNA expression.Western blot(WB)was used to detect the expressions of Wnt,β-catenin,CyclinD1,MDR1 and Bcl-2.Double luciferase activity experiment verified that miR-1180 targeted Wnt.Results The transfection efficiency of control group and silencing group were(93.51±1.89)%and(94.09±2.26)%,respectively.Compared with the blank group and the control group,the cell viability,IC_(50) value and apoptosis rate of the silencing group were decreased,and the differences were statistically significant(P<0.05),and there were no significant differences in cell viability,IC_(50) value and apoptosis rate between the blank group and the control group(P>0.05);Compared with the blank group and the control group,the mRNA and protein expressions of miR-1180,Wnt,β-catenin,CyclinD1,MDR1,Bcl-2 were decreased,and the differences were statisticall

关 键 词：微小核糖核酸-1180 结肠癌 奥沙利铂 化疗敏感性 

分 类 号：R735.3[医药卫生—肿瘤]