RNA激活上调p21^(WAF1/CIP1)基因表达对肺癌细胞增殖和凋亡影响的研究

Inhibition effect of up-regulation of p21^(WAF1/CIP1) by small activating RNA on cell proliferation and promotes apoptosis in lung cancer cells

目的:探讨应用RNA激活(RNAa)技术上调p21WAF1/CIP1(p21)表达对人肺癌H441细胞增殖和凋亡的影响。方法:设计靶向抑癌基因p21WAF1/CIP1启动子DNA序列互补的双链RNA分子(dsp21),转染H441细胞后应用半定量逆转录聚合酶链反应(RT-PCR)及蛋白质印迹法检测p21WAF1/CIP1基因mRNA及蛋白质的表达变化,噻唑蓝(MTT)法检测H441细胞增殖速度的变化,流式细胞仪检测H441细胞凋亡率的变化。结果:dsp21转染H441细胞72 h后p21WAF1/CIP1表达显著上调。RT-PCR灰度比值结果显示,空白对照组、阴性对照组和实验组p21WAF1/CIP1mRNA相对表达量分别为(38.1±2.5)%、(33.5±3.9)%和(96.5±2.3)%,差异有统计学意义,F=87.0,P<0.01;蛋白质印迹法结果显示,空白对照组、阴性对照组和实验组p21WAF1/CIP1蛋白相对表达量分别为(45.7±2.2)%、(43.2±3.1)%和(93.6±2.5)%,差异有统计学意义,F=79.0,P<0.01;经dsp21作用的H441细胞增殖受到明显抑制,凋亡率较对照组明显升高,F=10.2,P<0.01。结论:RNAa技术激活p21WAF1/CIP1基因表达并抑制肺癌H441细胞增殖,诱导细胞凋亡,为肺癌的基因治疗提供了新的思路和方法。

OBJECTIVE： To observe the effects of p21WAF1/CIP1 activation by RNAa on lung cancer cell proliferation and apoptosis.METHODS： A dsRNA（dsp21） targeting the p21WAF1/CIP1 gene promoter at position-322 relative to the transcription start site was transfected into H441 cells.Expression of mRNA and protein was evaluated by semiquantitative RT-PCR and western blot.The H441 cells proliferation was measured by MTT method,and the apoptosis rate was detected by flow-cytometry.RESULTS： The introduction of dsp21 was showed to efficiently upregulate the expression of p21WAF1/CIP1 gene according to the results of RT-PCR and wastern blot,the expressions of blank group,negative group and experiment group were（38.1±2.5）%,（33.5±3.9）% and（96.5±2.3）% by RFPCR（F=87.0,P0.01）,and（45.7±2.2）%,（43.2±3.1）% and（93.6±2.5）% in western blot（F=79.0,P0.01,as compared with controls）.The inhibitory effect on cell proliferation was confirmed by MTT test（P0.05,as compared with controls）.Apoptosis rate of H441 cells treated with dsp21 was significantly higher than that of the control cells（F=10.2,P0.01）.CONCLUSION： dsp21 targeting p21WAF1/CIP1 gene promoter can specifically upregulate the expression of p21WAF1/CIP1 gene in H441 cells,and therefore has an substantially inhibitory effect on cell proliferation in vitro,it provides a new method and material to the gene therapy of lung cancer.