G2/M期P53/CDK1通路对卵巢癌细胞增殖和凋亡的影响

Roles of P53/CDK1 pathway of G2/M checkpoint on proliferation and apoptosis of ovarian cancer cells

目的探讨细胞周期G2/M期P53-P21WAF1-细胞周期依赖激酶1(cyclin dependent kinases 1,CDK1)通路与上皮性卵巢癌细胞增殖和凋亡的关系。方法分别将CDK1、p53基因特异性shRNA质粒及阴性对照质粒转染卵巢上皮性癌SK-OV-3、OVCAR-3细胞,MTT法、TUNEL法和FCM法分别检测细胞增殖、凋亡和细胞周期分布情况,Western blot法分别检测各组细胞中CDK1、磷酸化CDK1(Thr14/Tyr15)、P53、p-P53(ser15)、P21WAF1、Cyclin B1以及增殖和凋亡相关蛋白Ki-67、Survivin、Caspase3蛋白的表达。结果分别沉默CDK1、p53基因后,SK-OV-3和OVCAR-3细胞增殖受到抑制、凋亡增加、G2期细胞数目增加;Ki-67、Survivin蛋白表达减少,cleaved-Caspase3蛋白表达增加。此外,沉默p53基因可引起p-P53(ser15)和P21WAF1蛋白表达减少、p-CDK1(Thr14/Tyr15)蛋白表达升高。结论 G2/M检验点P53/CDK1信号通路可能参与卵巢癌上皮性细胞增殖和凋亡的调节。

Objective To explore the role of P53-P21 WAF1-cyclin dependent kinases 1（ CDK1） pathway of cell cycle G2/M checkpoint on the proliferation and apoptosis of ovarian cancer cells. Methods The specific short-hair RNA（ shRNA） plasmids and negative control plasmid of CDK1 and p53 genes were transfected into SK-OV-3 and OVCAR-3 cells respectively. Cell proliferation,cell apoptosis rate and cell cycle distribution were measured by MTT,TUNEL and FCM assay respectively. Expressions of CDK1,phospho-CDK1（ Thr14/Tyr15）,P53,p-P53（ ser15）,P21 WAF1,Cyclin B1,Ki-67,Survivin and cleaved-Caspase3 proteins were examined by Western blot.Results After silencing the CDK1 and p53 genes by RNAi respectively,the proliferation of SK-OV-3 and OVCAR-3 cells were inhibited,the apoptosis increased and the number of G2 phase cells increased,expressions of Ki-67 and Survivin protein were downregulated,expressions of cleaved-Caspase3 were upregulated. Moreover,the levels of p-P53（ ser15） and P21 WAF1 were decreased,p-CDK1（ Thr14/Tyr15） were increased after p53 gene knockdown. Conclusion P53/CDK1 pathway of G2/M checkpoint may be implicated in the proliferation and apoptosis regulation of ovarian cancer cells.