RNAi沉默PELPI/MNAR基因对子宫内膜癌细胞增殖和细胞周期的作用

Effects of Silencing PELP1 / MNAR Expression by RNA Interference on Proliferation and Cell Cycle of Endometrial Cancer Cells

【目的】构建脯氨酸-谷氨酸-亮氨酸富集蛋白1(PELP1)基因慢病毒表达载体,研究沉默PELP1/雌激素受体非基因组活性辅助调节因子(PELP1/MNAR)基因表达对子宫内膜癌细胞增殖和周期的作用及机制。【方法】构建合成靶向PELP1/MNAR RNAi慢病毒表达载体,并转染子宫内膜癌Ishikawa细胞,Real-time PCR和western blot检测PELP1/MNAR mRNA和蛋白水平的表达。使用普通培养基和加入E2的培养基培养各组细胞,MTT检测各组细胞增殖情况;流式细胞仪检测细胞生长周期情况;Western blot检测ER下游靶基因c-fos,cyclin D1的蛋白表达水平。【结果】成功构建合成靶向PELP1/MNAR RNAi慢病毒表达载体,转染子宫内膜癌Ishikawa细胞,转染后PELP1/MNAR mRNA的表达和蛋白的表达分别下降86%和65%(P<0.05)。转染后Ishikawa细胞与对照组相比增殖抑制(P<0.05),细胞周期G0/G1期细胞比例增加,S期细胞比例减少(P<0.05)。加入雌激素后,三组细胞的生长速度加快,细胞S期的比例增加,转染组增殖抑制明显(P<0.05),S期比例降低(P<0.05)。转染组ER下游基因c-fos、cyclin-D1蛋白水平均显著降低(P<0.05)。【结论】在子宫内膜癌细胞中沉默PELP1/MNAR的表达能能抑制细胞生长、使细胞阻滞在G1期,下调ER靶基因蛋白表达,PELP1/MNAR有望成为子宫内膜癌治疗的潜在靶点。

[Objective] To establish a lentiviral vector based RNA interference expression system targeting PELP1/MNAR to obtain stable transcript knockdown,and investigate the effect of down-regulation of PELP1/MNAR on proliferation,cell cycle of endometrial cancer cell with or without estrogen stimulation,and its mechanisms.[Methods] The shRNA oligonueleotides targeting to PELP1/MNAR gene were synthesized and cloned to generate shRNA lentivirus expressive vectors.Endometrial cancer cell Ishikawa was transfected,and gene silencing effect was determined by real-time PCR and Western blot analysis on the level of mRNA and protein.With or without E2 stimulation,the proliferation rate of transfected Ishikawa cells were detected by MTT; cell cycle were measured by flow cytometry.The protein expression of the ER target gene c-fos,cyclin D1 were detected by Western blot.[Result]The RNAi lentivirus expression vector targeting PELP1/MNAR sequence were constructed and the endometrial cancer cell Ishikawa was transfected successfully.Compared with control,the levels of PELP1/MNAR mRNA and protein in transfected Ishikawa cells were significantly reduced by 86 and 65%,respectively （P 〈 0.05）.The transfected Ishikawa cells showed a decrease in proliferation compared with the parental cells and that the effect of PELP1/MNAR down-regulation on cell proliferation was more pronounced when 17β-E2 was added （P 〈 0.05）.The results of flow cytometry showed a clear effect of transfected Ishikawa cells were arrested on the G0/G1 phase of the cells compared with the control with or without E2 treatment.Transfected Ishikawa cells were reduced sharply in S phrase,accordingly （P 〈 0.05）.The down-regulation of PELP1/MNAR in Ishikawa cells also reduced expression of the ER target gene c-fos,cyclin-D1.[Conclusion] PELP1/MNAR down-regulation substantially reduced cell proliferation and G1-S cell cycle progression of Ishikawa cells with or without E2 stimulation.The silencing effect of PELP1/MNAR down regulate the expression of ER target gene c-fos,cyclin D1.PELP1/MNAR might be a potential therapeutic target for endometrial cancer.