microRNA-21靶向调控Wnt2基因对肝癌细胞HepG2增殖与迁移的影响  

Effect of targeted regulation of the Wnt2 gene by microRNA(microRNA-21)on the proliferation and migration of HepG2 hepatoma cells

作　　者：王泽鑫[1] 关利君[1] 李建明[1] 王志超 薛梦若 秦孝军[1] WANG Zexin;GUAN Lijun;LI Jianming(Department of Interventional Radiology, The Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010050, China)

机构地区：[1]内蒙古医科大学附属医院介入放射科,呼和浩特010050

出　　处：《临床肝胆病杂志》2020年第4期803-807,共5页Journal of Clinical Hepatology

基　　金：内蒙古自治区自然科学基金(2017MS0890)。

摘　　要：目的探讨分析microRNA-21(miR-21)靶向调控Wnt2基因对肝癌细胞HepG2增殖与迁移的影响。方法采用实时荧光定量PCR法检测肝癌细胞HepG2及正常肝细胞株LO2 miR-21的表达情况。对HepG2细胞转染miR-21抑制剂,分析转染后抑制剂组与对照组miR-21的表达情况、细胞增殖、迁移及凋亡情况,检测两组细胞Wnt2蛋白表达水平,并采用双荧光素酶报告基因实验验证miR-21与Wnt2基因的关系。计量资料两组间比较采用t检验。结果HepG2细胞miR-21相对表达水平明显高于LO2细胞(1.978±0.035 vs 1.586±0.022,t=16.424,P<0.05)。转染miR-21抑制剂后,抑制剂组miR-21相对表达水平较对照组显著降低(0.857±0.017 vs 1.684±0.039,t=33.669,P<0.05)。转染miR-21抑制剂24、48、72 h后,抑制剂组HepG2细胞增殖能力均较对照组显著降低(P值均<0.05);抑制剂组穿过Tranwell小室的细胞数显著低于对照组(83.72±15.06 vs 147.85±20.64,t=4.347,P<0.05);抑制剂组细胞凋亡率明显高于对照组(25.67%±3.95%vs 10.27%±2.14%,t=5.937,P<0.05)。抑制剂组HepG2细胞的Wnt2相对表达水平明显低于对照组(0.862±0.127 vs 1.306±0.218,t=3.048,P<0.05)。TargetScan软件显示,miR-21抑制剂可显著抑制野生型Wnt2-3′UTR质粒转染细胞的荧光素酶活性(0.972±0.102 vs 0.612±0.092,t=4.219,P<0.05),而对突变型Wnt2-3′UTR质粒转染细胞的荧光素酶活性并无明显影响(0.982±0.093 vs 0.911±0.128,t=0.972,P>0.05)。结论抑制miR-21表达可有效抑制HepG2细胞增殖和迁移,促进HepG2细胞凋亡,并抑制Wnt信号通路的过度激活,可能成为肝癌治疗的潜在靶基因之一。Objective To investigate the effect of targeted regulation of the Wnt2 gene by microRNA(miR-21)on the proliferation and migration of HepG2 hepatoma cells.Methods Quantitative real-time PCR was used to measure the mRNA expression of miR-21 in HepG2 hepatoma cells and normal liver cell line LO2.HepG2 cells were transfected with miR-21 inhibitor,and then the expression of miR-21 and cell proliferation,migration,and apoptosis were analyzed for the inhibitor group and the control group.The protein expression of Wnt2 was measured for the two groups,and dual-luciferase reporter assay was used to verify the association between miR-21 and the Wnt2 gene.The t-test was used for comparison of continuous data between groups.Results The relative expression of miR-21 in HepG2 cells was significantly higher than that in LO2 cells(1.978±0.035 vs 1.586±0.022,t=16.424,P<0.05).After the transfection of miR-21 inhibitor,the inhibitor group had significantly lower expression of miR-21 than the control group(0.857±0.017 vs 1.684±0.039,t=33.669,P<0.05).Compared with the control group after the transfection of miR-21 inhibitor,the inhibitor group had a significant reduction in the proliferation ability of HepG2 cells(P<0.05),a significantly lower number of cells passing through the Transwell chamber(83.72±15.06 vs 147.85±20.64,t=4.347,P<0.05),and a significantly higher cell apoptosis rate(25.67%±3.95%vs 10.27%±2.14%,t=5.937,P<0.05).The inhibitor group had significantly lower relative expression of Wnt2 in HepG2 cells than the control group(0.862±0.127 vs 1.306±0.218,t=3.048,P<0.05).TargetScan software showed that miR-21 inhibitor significantly inhibited the luciferase activity of the cells transfected with wild-type Wnt2-3′UTR plasmid(0.972±0.102 vs 0.612±0.092,t=4.219,P<0.05),while there was no effect on the luciferase activity of the cells transfected with mutant Wnt2-3′UTR plasmid(0.982±0.093 vs 0.911±0.128,t=0.972,P>0.05).Conclusion Inhibition of miR-21 expression can effectively inhibit the proliferation and migrat

关 键 词：微RNAS Wnt2蛋白质 癌 肝细胞 细胞增殖 细胞运动 

分 类 号：R735.7[医药卫生—肿瘤]