RNA干扰抑制HMGB1基因表达对子宫内膜癌的增殖抑制作用及其分子机制

Inhibition on the proliferation of human endometrial cancer cells by RNAi inhibiting HMGB1 gene expression and its possible molecular mechanism

目的:研究RNA干扰抑制高迁移率族蛋白1(high mobility group box-1 protein,HMGB1)的表达对子宫内膜癌细胞株HEC-1A的增殖抑制作用及对细胞周期、细胞凋亡的影响,并探讨其可能的分子机制。方法:构建靶向HMGB1基因的慢病毒shRNA载体,转染子宫内膜癌细胞株HEC-1A,同时利用实时荧光定量RT-PCR和Western印迹检测转染HMGB1-siRNA后细胞HMGB1 mRNA和蛋白表达水平的变化。MTT法和流式细胞术分别检测转染HMGB1-siRNA后HEC-1A细胞增殖、细胞周期和细胞凋亡的变化。Western印迹检测转染HMGB1-siRNA后细胞AKT,pAKT和cyclinD1的蛋白表达水平。结果:慢病毒HMGB1 shRNA载体成功抑制HMGB1 mRNA(P<0.05)和蛋白(P<0.01)在HEC-1A细胞中的表达;转染HMGB1 shRNA后HEC-1A细胞的增殖能力明显受抑制(P<0.01),并能阻滞细胞于G0/G1期,且明显诱导细胞凋亡;转染HMGB1 shRNA组、空白对照组和阴性对照组的总凋亡率分别为(17.89±0.23)%,(4.69±0.20)%和(4.62±0.17)%,3组之间差异有统计表达学意义(P<0.01)。转染HMGB1 shRNA后细胞pAKT和cyclinD1蛋白表达水平均下调(均P<0.01)。结论:HMGB1表达下调可以有效地抑制子宫内膜癌细胞HEC-1A的增殖,细胞阻滞于G0/G1期,且可诱导细胞凋亡,影响磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/AKT)信号通路及下调cyclinD1的蛋白表达水平可能为其主要作用机制。

Objective: To investigate the effect of HMGB1 small interfering RNA(siRNA) on the proliferation, cell cycle and apoptosis of human endometrial cancer cell line HEC-1A, and its possible molecular mechanism.Methods: Lentivirus vector with HMGB1 shRNA was constructed and infected the endometrial cancer cell line HEC-1A. After viral infection for 72 h, real time PCR and Western blot were performed to investigate HMGB1 mRNA and protein expression. The cell proliferation was determined with methyl thiazolyl tetrazolium(MTT) method. Flow cytometry was performed to analyze the cell cycle progression of propidium iodide(PI)-stained HEC-1A cells and the apoptotic rate of annexinV/PI-stained cells. Western blot was used to detect the protein expression of AKT, pAKT and CyclinD1.Results: Lentivirus vector with HMGB1 shRNA inhibited the mRNA(P<0.05) and protein(P<0.01) expression of HMGB1 in the cell line HEC-1A. The MTT assay demonstrated that HMGB1 knockdown significantly reduced the cell proliferation. FCM results showed that HMGB1 knockdown significantly resulted in the disruption of the cell cycle at G0/G1 phase and the induction of apoptosis. The apoptotic rate was(17.89±0.23)%,(4.69±0.20)% and(4.62±0.17)% in the HMGB1 knockdown group, the blank group and the negative group respectively. There was significance difference between the 3 groups(P<0.01). The protein expressions of pAKT and cyclinD1 were down-regulated after the HMGB1 knockdown for 72 h.Conclusion: Knockdown of HMGB1 expression can significantly inhibit the proliferation and induce the cell cycle arrest and apoptosis in the endometrial cancer cell line HEC-1A. PI3K/AKT pathway and down-regulation of the protein expression of cyclinD1 may be involved in its therapeutic mechanism.