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

摘要:

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siRNA和阴性对照序列转染喉癌Hep-2细胞，实验分为CIP2A siRNA组与Control siRNA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 mRNA表达水平，Western blot检测CIP2A及生长相关蛋白c-myc、AKT、p-AKT、cyclinD1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 siRNA组为339.00±34.00，CIP2A siRNA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 siRNA组IC50为（6.67±0.54） ?滋g/ml，CIP2A siRNA组为（3.53 ±0.32） ?滋g/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 siRNA组G1期细胞比例为66.860±1.972，CIP2A siRNA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 siRNA组为23.713±1.564，CIP2A siRNA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CyclinD1（t=-4.531，P=0.011）、c-myc（t=-4.522，P=0.011）、p-AKT（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、cyclinD1的表达有关。

Abstract:

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：siRNA-mediated CIP2A and negative control sequences were performed in Hep-2 cells. The experiment was designed into CIP2A siRNA group and control siRNA group. Colony formation assay was used to examine the role of CIP2A in cell proliferation. The impact of CIP2A 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 CIP2A，c-myc，AKT，p-AKT，and cyclinD1. Results：CIP2A knockdown in Hep-2 cells markedly decreased both CIP2A mRNA 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，CIP2A depletion blocked cell cycle at G1 phase（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 CIP2A depletion downregulated CyclinD1，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 cyclinD1.