肺癌细胞中survivin与P53的相关性

Relationship between P53 and survivin in human lung cancer cells

目的:观察survivin特异性小干扰RNA(small interference RNA,siRNA)对P53野生型和P53缺失型肺癌细胞株的生长抑制作用,分析肺癌细胞中survivin与P53的相关性。方法:以化学合成的survivin siRNA转染P53野生型肺癌细胞株A549和P53缺失型肺癌细胞株H1299,用MTT法检测转染前后肺癌细胞的增殖情况,流式细胞术分析细胞周期和凋亡的改变,半定量RT-PCR检测转染对survivin mRNA表达的影响,Western blotting检测survivin、PARP、P21、P53等蛋白表达的变化。结果:siRNA转染前A549细胞中survivin表达量明显低于H1299细胞(P<0.05)。Survivin siRNA转染组两种细胞survivin蛋白及RNA表达水平均显著低于对照组和无关干扰组(P<0.01)。Survivin siRNA对A549和H1299细胞生长抑制作用均呈浓度依赖性(P<0.05),但对H1299细胞的抑制起效晚于A549,且抑制程度低于A549细胞。转染后A549和H1299细胞均有G_1期比例增加和S期比例相应地减少,H1299细胞的变化更为显著。A549细胞有一定比例出现凋亡,PARP发生了裂解,P53、P21蛋白表达增加,而H1299细胞中上述指标的变化却不明显。结论:P53野生型A549细胞内源性survivin表达明显低于P53缺失型H1299细胞;抑制survivin能上调P53蛋白表达,其对A549细胞的生长、凋亡和P21等蛋白表达的影响明显强于H1299细胞;提示survivin与P53之间可能存在着相互抑制作用。

Objective：To observe the inhibitory effects of survivin-specific siRNA on human lung cancer cell lines with different P53 statuses, and to analyse the relationship between P53 and survivin in vitro. Methods： A549 （wild-type P53） and H1299 （P53 absent） cells were transfected with chemosynthetic survivin siRNA. The effect of siRNA on cell proliferation was analysed by MTT assay. The changes of cell cycle and apoptosis were examined by flow cytometry. The expression of survivin mRNA was detected by RT-PCR. The protein expression of survivin, P53, P21 and PARP was examined by Western blotting. Results： Endogenous survivin level in A549 cells was lower than that in H1299 cells. The mRNA and protein expression of survivin was significantly lower in siRNA group than in blank control group and non-silencing siRNA group （P 〈0.01 ）. Survivin-specific siRNA significantly inhibited the gyowth and proliferation of both A549 and H1299 cells in a concentration dependent manner （P 〈 0.05）, but the inhibitory effect was earlier and more efficient in A549 cells than in H1299 ceils. Survivin-specific siRNA produced an increase in the G1 fraction and a decrease in S fraction in the two cell lines, with the changes in the latter more evident. Apoptosis was found in A549 cells; PARP was cleaved and P21 and P53 protein was increased in A549 cells. The above changes were not obvious in H1299 cells. Conclusion： Endogenous survivin levels in A549 （wild-type P53 ） cells is lower than that in H1299 （without P53 ） cells. Inhibition of survivin by transfecting survivin siRNA can significantly up-regulate P53 protein expression in A549 cells. The effects of survivin siRNA on the cell growth, apoptosis and expression of some proteins are more prominent in A549 than in H1299 cells, suggesting that survivin and P53 might inhibit each other.