论文部分内容阅读
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。
AIM: To investigate the effects of up-regulation of p21WAF1 / CIP1 (p21) expression on the proliferation and apoptosis of human lung cancer H441 cells by RNAi (RNAi) technology. Methods: Double-stranded RNA (dsp21) targeting the tumor suppressor gene p21WAF1 / CIP1 promoter was designed and transfected into H441 cells. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression of p21WAF1 / CIP1 mRNA and protein expression were detected. The proliferation of H441 cells was detected by MTT assay. The apoptosis rate of H441 cells was detected by flow cytometry. Results: The expression of p21WAF1 / CIP1 was significantly up-regulated after transfected into H441 cells by dsp21 for 72 h. RT-PCR results showed that the relative expression levels of p21WAF1 / CIP1 mRNA in the blank control group, negative control group and experimental group were (38.1 ± 2.5)%, (33.5 ± 3.9)% and (96.5 ± 2.3)%, respectively The results of Western blotting showed that the relative expression levels of p21WAF1 / CIP1 protein in the blank control group, negative control group and experimental group were (45.7 ± 2.2)% and (43.2 ± 3.1)%, respectively (93.6 ± 2.5)%, the difference was statistically significant (F = 79.0, P <0.01). The proliferation of H441 cells treated with dsp21 was significantly inhibited and the apoptosis rate was significantly higher than that of the control group (F = 10.2, P <0.01) . Conclusion: RNAi can activate p21WAF1 / CIP1 gene expression and inhibit the proliferation and induce apoptosis of lung cancer H441 cells. It provides a new way of gene therapy for lung cancer.