蛋白磷酸酶2A的癌性抑制因子表达对喉癌细胞株Hep-2增殖的影响及相关机制研究

Effects of cancerous inhibitor of protein phosphatase 2A expression on proliferation of laryngeal squamous cell carcinomas cell Hep-2 and its mechanism

目的:研究蛋白磷酸酶2A的癌性抑制因子(cancerous inhibitor of protein phosphatase 2A,CIP2A)表达对喉癌细胞株Hep-2增殖的影响及相关分子机制。方法:以特异性si RNA和阴性对照序列转染喉癌Hep-2细胞,实验分为CIP2A si RNA组与Control si RNA组。平板克隆形成实验检测CIP2A在喉癌细胞增殖中的作用;MTT法检测CIP2A对细胞生长曲线及顺铂敏感性影响;流式细胞术检测细胞周期分布和凋亡率的改变。real-time PCR检测CIP2A m RNA表达水平,Western blot检测CIP2A及生长相关蛋白c-myc、AKT、p-AKT、cyclin D1的蛋白表达水平。结果:沉默CIP2A后,CIP2A在基因及蛋白水平的表达量明显降低,Hep-2细胞生长减缓,克隆的形成被抑制,Control si RNA组为339.00±34.00,CIP2A si RNA组为126.00±33.00,差异有统计学意义(t=-7.796,P=0.010);沉默CIP2A可增加喉癌细胞对顺铂的敏感性,Control si RNA组IC50为(6.67±0.54)μg/ml,CIP2A si RNA组为(3.53±0.32)μg/ml,差异有统计学意义(t=8.598,P=0.001);细胞周期阻滞于G1期,Control si RNA组G1期细胞比例为66.860±1.972,CIP2A si RNA组为74.613±3.357,差异有统计学意义(t=3.449,P=0.026);S期细胞比例减少,Control si RNA组为23.713±1.564,CIP2A si RNA组为17.747±1.255,差异有统计学意义(t=-5.153,P=0.007);沉默CIP2A没有引起细胞凋亡率的变化(P=0.216)。Western blot结果显示,沉默CIP2A表达可下调Cyclin D1(t=-4.531,P=0.011)、c-myc(t=-4.522,P=0.011)、pAKT(t=-4.574,P=0.010)的表达,但对总AKT蛋白的表达没有影响(t=0.051,P=0.962)。结论:沉默CIP2A基因可抑制喉癌Hep-2细胞生长,增加喉癌细胞对顺铂的敏感性,其机制可能与下调c-myc、p-AKT、cyclin D1的表达有关。

Objective：To investigate the effects of cancerous inhibitor of protein phosphatase 2A（CIP2A）expression on proliferation of laryngeal squamous cell carcinomas cells Hep-2 and its mechanism. Methods：si RNA-mediated CIP2 A and negative control sequences were performed in Hep-2 cells. The experiment was designed into CIP2 A si RNA group and control si RNA group. Colony formation assay was used to examine the role of CIP2 A in cell proliferation. The impact of CIP2 A on cell growth and cisplatin sensitivity was evaluated using MTT assay. Flow cytometry was used to examine cell cycle distribution and apoptotic rate. Quantitative real-time PCR and Western blot were applied to assess the expression of CIP2 A,c-myc,AKT,p-AKT,and cyclin D1. Results：CIP2A knockdown in Hep-2 cells markedly decreased both CIP2 A m RNA and protein expression,reduced the proliferation speed and colony formation（P=0.010）and increased the drug sensitivity of the cells to cisplatin（P=0.001）. In addition,CIP2 A depletion blocked cell cycle at G1phase（P=0.026）and decreased the percentage of S stage cells（P=0.007）,and did not significantly alter cell apoptosis rate（P=0.216）.Western blot showed that CIP2 A depletion downregulated Cyclin D1,c-myc,p-AKT expression（P=0.011,P=0.011,P=0.010）,but had no effects on the expression of total AKT protein（P =0.962）.Conclusion：CIP2A depletion inhibits cell proliferation and increases the sensitivity to cisplatin,mechanism of which may be related with decreased c-myc,p-AKT,and cyclin D1.