微RNA-125b靶向调控M2型丙酮酸激酶影响宫颈癌SiHa细胞机制验证

目的探讨宫颈癌细胞中微RNA(miR)-125b和M2型丙酮酸激酶(PKM2)的表达以及miR-125b对PKM2基因的调控作用,从而确定miR-125b下游调控的靶基因,阐明其在宫颈癌发生发展中可能的分子机制。方法培养宫颈癌SiHa细胞及正常上皮细胞(293T细胞),利用细胞转染技术过表达或沉默miR-125b。实验分为实验组:miR-125b mimics(M组)、miR-125b inhibitor(I组);阴性对照组:miR-125b mimics negative con-trol(MNC组)、miR-125b inhibitor negative control(INC组);空白对照组(B组):未进行转染。采用实时荧光定量-聚合酶链反应(qRT-PCR)检测各组细胞中miR-125b的含量。采用免疫荧光及蛋白质印迹法检测各组细胞中PKM2蛋白的表达。结果与293T细胞相比,宫颈癌SiHa细胞中miR-125b的表达下调(0.82±0.05)(P<0.05),PKM2表达上调(3.4±0.5)倍(P<0.05)。SiHa细胞转染成功后,免疫荧光检测miR-125b模拟物组表达PKM2蛋白的细胞数目(1.0±0.7)低于模拟物阴性对照组表达PKM2蛋白的细胞数目(3.6±0.5)(P<0.05);miR-125b抑制物组中表达PKM2蛋白的细胞数目(6.0±0.7)高于抑制物阴性对照组表达PKM2蛋白的细胞数目(3.6±1.3)(P<0.05)。蛋白质印迹法检测miR-125b模拟物组的PKM2蛋白相对表达量(0.239±0.042)低于模拟物阴性对照组的PKM2蛋白相对表达量(0.314±0.018)(P<0.05);miR-125b抑制物组的PKM2蛋白表达水平(0.40±0.03)高于抑制物阴性对照组的PKM2蛋白表达水平(0.34±0.04)(P<0.05)。结论与正常上皮细胞相比,宫颈癌SiHa细胞中miR-125b表达下调,而PKM2蛋白表达上调;宫颈癌细胞中miR-125b表达下调可能会通过增加PKM2的表达,从而增强其糖酵解作用,促进肿瘤的生长。

Validating the mechanism underlying the effect of miR-125b targeted regulation of PKM2 on cervical cancer SiHa cells

Objective To investigate the expression of miR-125b and pyruvate kinase type M2(PKM2)in cervical cancer cells and the regulatory effect of miR-125b on PKM2 gene,so as to determine the target genes for downstream regulation of miR-125b and clarify on the possible molecular mechanism underlying its role in development of cervical cancers.Methods SiHa cells and normal epithelial cells(293T cells)were cultured.MiR-125b was overexpressed or silenced via cell transfection.In this study,the experimental groups were miR-125b mimics group(M group)and miR-125b inhibitor group(Group I),and the negative control groups were miR-125b mimics negative control group(MNC group)and miR-125b inhibitor negative control group(INC group).An untransfected blank control group(group B)was also set.The levels of miR-125b in each group of cells were detected by qRT-PCR.Immunofluorescence and Western blotting were used to detect the expression of PKM2 protein in each group of cells.Results Compared with 293T cells,the expression of miR-125b was down-regulated by(0.82±0.05)folds(P<0.05),and that of PKM2 was up-regulated by(3.4±0.5)folds(P<0.05),in cervical cancer SiHa cells.After successful transfection of SiHa cells,the number of cells expressing PKM2 protein in the miR-125b mimic group(1.0±0.7)was lower than that in the mimics negative control group(3.6±0.5)(P<0.05),and the number of cells expressing PKM2 protein in the miR-125b inhibitor group(6.0±0.7)was higher than that in the inhibitor negative control group(3.6±1.3)(P<0.05).Western blotting showed that the relative expression level of PKM2 protein in miR-125b mimic group(0.239±0.042)was lower than that in the mimics negative control group(0.314±0.018)(P<0.05).The expression level of PKM2 protein in the miR-125 inhibitor group(0.40±0.03)was higher than that in the inhibitor negative control group(0.34±0.04)(P<0.05).Conclusion Compared with normal epithelial cells,the miR-125b expression is down-regulated,and the PKM2 protein expression is up-regulated,in cervical cancer SiHa cells.The down-regulation of miR-125b in cervical cancer cells may increase the expression of PKM2,enhance its glycolysis and thereby promote tumor growth.