RNA干扰抑制高迁移率族蛋白1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和细胞凋亡的影响

Inhibition of high mobility group box-1 protein expression by RNA interference inhibits the proliferation of endometrial carcinoma cell line HEC-1A and its effect on cell cycle and apoptosis

目的探讨通过RNA干扰阻滞高迁移率族蛋白1(HMGB1)的表达水平对子宫内膜癌(EC)细胞株HEC-1A中的细胞增殖、细胞周期及细胞凋亡的影响。方法将细胞分为以下三组:空白对照组(HEC-1A细胞未感染)、阴性对照组(HEC-1A细胞感染无关序列)、干预组(慢病毒转染HEC-1A细胞)。通过Western blot法对三组HEC-1A细胞中的HMGB1蛋白表达水平进行检测,并采用实时荧光定量PCR对HMGB1 mRNA相对表达量进行检测。采用MTT法对HEC-1A细胞增殖情况进行检测,用流式细胞术对HEC-1A细胞周期变化进行检测,同时用Annexin VFITC/PI法对HEC-1A细胞凋亡情况进行检测。采用Western blot法对转染HMGB1-siRNA后细胞cyclin D1、丝氨酸/苏氨酸蛋白激酶(Akt)及磷酸化Akt(p Akt)蛋白表达进行检测。结果相比空白对照组与阴性对照组,干预组HEC-1A细胞中HMGB1蛋白表达水平显著下降(P <0. 01)。经实时荧光定量PCR检测结果可知相比空白对照组与阴性对照组,干预组HEC-1A细胞中HMGB1 mRNA相对表达量显著下降(P <0. 01)。相比空白对照组、阴性对照组,干预组cyclin D1和p Akt蛋白表达显著下降(P <0. 01);而三组Akt蛋白表达水平的比较,并无显著差异(P> 0. 05)。相比空白对照组、阴性对照组,干预组HEC-1A细胞在570 nm波长处的吸光度值显著下降(P <0. 01)。干预组HEC-1A细胞G0/G1期比率在整个细胞周期分布中的比率最高,其较空白对照组、阴性对照组显著升高(P <0. 01);干预组HEC-1A细胞S期比率、G2/M期比率较空白对照组、阴性对照组显著下降(P <0. 01)。干预组HEC-1A细胞凋亡比率较空白对照组、阴性对照组显著升高(P <0. 01)。结论 HMGB1表达降低可有效阻滞EC细胞株HEC-1A的增殖,使得细胞于G0/G1期受阻,同时可有效促进细胞凋亡,其作用机制可能在于降低cyclin D1蛋白表达和干扰磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)信号通路。

Objective To investigate the effects of blocking the expression of high mobility group box-1 protein (HMGB1) by RNA interference on cell proliferation, cell cycle and apoptosis in endometrial carcinoma (EC) cell line HEC-1A. Methods Cells were divided into three groups: blank control group (HEC-1A cells were not infected), negative control group (HEC-1A cell infection independent sequence), intervention group (lentivirus transfected HEC-1A cells). Western blot was used to detect the expression of HMGB1 in three groups of HEC-1A cells, and real-time fluorescence quantitative PCR was used to detect the relative expression of HMGB1. MTT assay was used to detect the proliferation of HEC-1A cells, flow cytometry was used to detect the cell cycle changes of HEC-1A cells, and Annexin V-FITC/PI method was used to detect the apoptosis of HEC-1A cells. The expression of cyclin D1, serine/threonine protein kinase (Akt) and phosphorylated Akt (pAkt) in HMGB1-siRNA transfected cells were detected by Western blot. Results Compared with blank control group and negative control group, the expression of HMGB1 protein in HEC-1A cells in intervention group was significantly decreased ( P <0.01). The results of real-time fluorescence quantitative PCR showed that the relative expression of HMGB1 in HEC-1A cells of intervention group was significantly lower than that of blank control group and negative control group ( P <0.01). Compared with the blank control group and the negative control group, the expression of cyclin D1 and pAkt protein in the intervention group decreased significantly ( P <0.01), while there was no significant difference in the expression level of Akt protein among the three groups ( P >0.05). Compared with blank control group and negative control group, the absorbance of HEC-1A cells in intervention group decreased significantly at 570 nm ( P <0.01). The proportion of G0/G1 phase of HEC-1A cells in intervention group was the highest in the whole cell cycle distribution, which was significantly higher than that of blank control group and negative control group ( P <0.01);the proportion of S phase and G2/M phase of HEC-1A cells in intervention group was significantly lower than that of blank control group and negative control group ( P <0.01). Compared with blank control group and negative control group, the apoptotic rate of HEC-1A cells in intervention group was significantly higher ( P <0.01). Conclusion Decreased expression of HMGB1 can effectively block the proliferation of EC cell line HEC-1A, block the cell at G0/G1 phase, and promote cell apoptosis. Its mechanism may be to reduce the expression of cyclinD1 protein and interfere with the signal pathway of phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt).