miR-196b通过LRIG2调控宫颈癌细胞增殖和凋亡生物学特性的分子机制

Molecular mechanism of miR-196b regulating the biological characteristics of proliferation and apoptosis in cervical cancer cells through LRIG2

目的:探讨miR-196b对宫颈癌细胞增殖和凋亡的影响以及潜在的作用机制。方法:运用实时定量聚合酶链式反应(qRT-PCR)检测宫颈癌细胞HeLa、SiHa、C33a、Caski和正常宫颈细胞株Ect1/E6E7中miR-196b和LRIG2 mRNA的表达水平;将宫颈癌SiHa细胞分为miR-con组(转染miR-con)、miR-196b组(转染miR-196b mimics)、anti-miR-con组(转染anti-miR-con)、anti-miR-196b组(转染miR-196b inhibitor)、miR-196b+si-con组(共转染miR-196b和si-con)、miR-196b+si-LRIG2组(共转染miR-196b和si-LRIG2)、miR-con+WT-LRIG2组(共转染miR-con和WT-LRIG2)、miR-con+MUT-LRIG2组(共转染miR-con和MUT-LRIG2)、miR-196b+WT-LRIG2组(共转染miR-196b和WT-LRIG2)、miR-196b+MUT-LRIG2组(共转染miR-196b和MUT-LRIG2)。采用MTT法检测细胞增殖;流式细胞术检测细胞凋亡;Western Blot检测蛋白表达;双荧光素酶报告实验检测荧光活性。结果:相较于正常宫颈细胞株Ect1/E6E7,宫颈癌细胞HeLa、SiHa、C33a、Caski中miR-196b的表达水平显著升高,LRIG2的表达水平显著降低(P<0.05);干扰miR-196b可抑制SiHa细胞增殖促进细胞凋亡;抑制Bcl-2、CDK4和Cyclin D1蛋白的表达,促进Bax、Caspase-3蛋白的表达。miR-196b靶向调控LRIG2的表达。抑制LRIG2表达能逆转干扰miR-196b对SiHa细胞的抑制增殖、促进凋亡的作用以及对AKT信号通路中PI3K、p-AKT、p-mTOR蛋白表达的抑制作用。结论:miR-196b可抑制宫颈癌细胞增殖,促进其凋亡,其机制可能与LRIG2及AKT信号通路有关,将可为宫颈癌的预防和治疗提供新靶点。

Objective:To investigate the effects of miR-196b on proliferation and apoptosis of cervical cancer cells and its potential mechanism.Methods:Expressions of of miR-196b and LRIG2 mRNA in cervical cancer cells HeLa,SiHa,C33a,Caski and normal cervical cell line Ect1/E6E7 were detected by real-time quantitative polymerase chain reaction(qRT-PCR).Cervical cancer SiHa cells were divided into miR-con group(transfected miR-con),miR-196b group(transfected miR-196b mimics),anti-miR-con group(transfected anti-miR-con),anti-miR-196b group(transfected anti-miR-196b),miR-196b+si-con group(co-transfected miR-196b and si-con),miR-196b+si-LRIG2 group(co-transfected miR-196b and si-LRIG2),miR-con+WT-LRIG2 group(co-transfected miR-con and WT-LRIG2),miR-con+MUT-LRIG2 group(co-transfected miR-con and MUT-LRIG2),miR-196b+WT-LRIG2(co-transfected miR-196b and WT-LRIG2),miR-196b+MUT-LRIG2 group(co-transfected miR-196b and MUT-LRIG2).Cell proliferation was detected by MTT assay;apoptosis was detected by flow cytometry;protein expression was detected by Western Blot;fluorescence activity was detected by double luciferase reporter assay.Results:Compared with the normal cervical cell line Ect1/E6E7,the expression levels of miR-196b in cervical cancer cells HeLa,SiHa,C33a and Caski were significantly increased,and the expression level of LRIG2 was significantly decreased(P<0.05);interference with miR-196b inhibited SiHa cell proliferation and promotes apoptosis;inhibited the expressions of Bcl-2,CDK4 and Cyclin D1 proteins,and promoted the expression of Bax and Caspase-3 proteins.miR-196b targets and regulate the expression of LRIG2.Overexpression of LRIG2 reversed the inhibitory effect of miR-1.