微小RNA-181b通过调控第10号染色体缺失的磷酸酶表达影响肝癌细胞的增殖与凋亡

Effect of microRNA-181 on proliferation and apoptosis of hepatocellular carcinoma cells via regulation of phosphatase and tensin ho-molog deleted from chromosome 10

目的探讨微小RNA(miRNA,miR)-181b通过调控人第10号染色体缺失的磷酸酶(PTEN)表达对HepG2肝癌细胞的增殖与凋亡的影响。方法利用脂质体Lipofectamine^TM 3000将miR-181b inhibitor和miR-181b NC转入HepG2细胞中,实时定量聚合酶链反应(Real-time PCR)检测miR-181b表达,噻唑蓝(MTT)法检测细胞活力,细胞克隆形成实验检测细胞克隆能力,流式细胞术检测细胞凋亡及细胞周期,Real-time PCR法及Western blot法检测细胞中PTEN mRNA及蛋白表达水平,并进行荧光素酶报告基因分析。结果miR-181b inhibitor组(0.23±0.02)中miR-181b表达量低于miR-181b NC组(1.00±0.01,P<0.05);miR-181b inhibitor组细胞活力(0.37±0.03)低于miR-181b NC组(0.59±0.05,P<0.05);miR-181b inhibitor组细胞克隆数目(37.64±3.70)低于miR-181 NC组(132.56±12.37,P<0.05);miR-181b inhibitor组细胞早期凋亡率和晚期凋亡率均高于miR-181b NC组(P<0.05)。miR-181b inhibitor组细胞周期G1期高于miR-181 NC组(P<0.05),miR-181b inhibitor组PTEN的mRNA及蛋白表达量均高于miR-181 NC组(P<0.05),且荧光素酶报告基因结果证实PTEN是miR-181b下游靶蛋白。结论miR-181b inhibitor通过负性调控PTEN表达抑制HepG2细胞增殖,并诱导细胞凋亡。

Objective To explore effect of microRNA (miRNA, miR)-181b on proliferation and apoptosis of hepatocellular carcinoma cells via regulation of phosphatase and tensin ho-molog deleted from chromosome 10 (PTEN). Methods miR-181b inhibitor and miR-181b NC were transfected into HepG2 cell by liposome Lipofectamine^TM 3000. The expression of miR-181b was detected by real-time quantitative polymerase chain reaction (Real-time PCR). Cell viability was measured by methyl thiazol tetrazolium (MTT) assay. Cell clone ability was detected by cell clone formation test. Cell apoptosis and cell cycle was detected by flow cytometry. The expression of PTEN protein and mRNA was measured by Western blotting and Real-time PCR. Luciferase reporter analysis was performed. Results The expression of miR-181b in miR-181b inhibitor group (0.23±0.02) was lower than that in miR-181b NC group (1.00±0.01, P<0.05). Cell viability in miR-181b inhibitor group (0.37±0.03) was lower than that in miR-181b NC group (0.59±0.06, P<0.05). Clone formation test showed cell clone number in miR-181b inhibitor group (37.64±3.70) was lower than that in miR-181b NC group (132.56±12.37, P<0.05). Cell early apoptotic rate and late apoptotic rate in miR-181b inhibitor group was higher than that in miR-181b NC group (P<0.05). At the same time, compared with miR-181b NC group, cell cycle was made arrested at G1 phase (P<0.05), the expression of PTEN protein and mRNA was up-regulated in miR-181b inhibitor group (P<0.05), miR-181b inhibitor targeted regulation the expression of PTEN. Conclusion miR-181b inhibitor could inhibit HepG2 cell proliferation and induce cell apoptosis by targeting up-regulation expression of PTEN.