下调Girdin基因表达对肺癌细胞增殖凋亡、免疫因子IL-8和TNF-α及顺铂化疗敏感性的研究

Effect of down-regulating Girdin gene expression on lung cancer cell proliferation and apoptosis,secretion of immune factors IL-8 and TNF-α and cisplatin chemosensitivity

目的:探讨下调微丝附着梁蛋白(Girdin)基因表达对肺癌细胞增殖凋亡、免疫因子IL-8和TNF-α及顺铂化疗敏感性的影响。方法:以人胚肺成纤维细胞MRC5作为对照细胞,RT-PCR及Western blot分别检测Girdin在PC9、SPC-A-1、H322、H1299、A549肺癌细胞中的表达; SPC-A-1细胞分为空白对照组、阴性对照组、Girdin-siRNA组、顺铂组和Girdin-siRNA+顺铂组,各组细胞培养48 h,Western blot检测Girdin-siRNA转染SPC-A-1细胞的效果; MTT法检测各组细胞活力;流式细胞术检测各组细胞凋亡率; RT-PCR检测IL-8和TNF-α的mRNA表达; Western blot检测增殖相关蛋白细胞增殖核抗原(PCNA)、凋亡相关蛋白含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)、Bcl-2相关X蛋白(Bax)及PI3K/AKT信号通路磷脂酰肌醇-3激酶(PI3K)和磷酸化的丝氨酸苏氨酸激酶(p-AKT)的蛋白表达。结果:肺癌细胞中Girdin的mRNA及蛋白表达均明显高于在MRC5细胞表达(P<0. 05); Girdin-siRNA转染SPC-A-1细胞后Girdin的表达显著降低(P<0. 05);与空白对照组比较,Girdin-siRNA组和顺铂组OD值及IL-8、TNF-α、PCNA、PI3K、p-AKT的表达均显著降低,细胞凋亡率及Caspase-3和Bax表达均显著升高(P<0. 05); Girdin-siRNA+顺铂组OD值及IL-8、TNF-α、PCNA、PI3K、p-AKT的表达均显著低于Girdin-siRNA组和顺铂组,细胞凋亡率及Caspase-3和Bax表达均显著高于Girdin-siRNA组和顺铂组(P<0. 05)。结论:抑制肺癌细胞Girdin表达可通过降低癌细胞增殖、诱导细胞凋亡和提高免疫增强乳腺癌顺铂化疗敏感性,对细胞增殖凋亡的机制可能与下调PI3K/AKT信号通路有关。

Objective:To investigate the effect of down-regulation of Girdin gene expression on proliferation,apoptosis,immune factors and cisplatin chemosensitivity of lung cancer cells.Methods:Human embryonic lung fibroblast MRC5 cells as control,RT-PCR and Western blot were used to detect the expression of Girdin in PC9,SPC-A-1,H322,H1299,A549 in lung cancer cells;SPC-A-1 cells were divided into blank control group,negative control group,Girdin-siRNA group,cisplatin group and Girdin-siRNA+cisplatin group,cells were cultured for 48 h,effect of Girdin-siRNA transfected SPC-A-1 cells were detected by Western blot;cell viability were detected by MTT;cell apoptosis was detected by flow cytometry;the expression of IL-8 and TNF-αmRNA were detected by RT-PCR;the expression of PCNA,Caspase-3,PI3K and p-AKT protein were detected by Western blot.Results:The mRNA and protein expression of Girdin in lung cancer cells were significantly higher than the expression in MRC5 cells(P<0.05);the expression of Girdin after Girdin-siRNA transfected SPC-A-1 cells was significantly decreased(P<0.05);compared with the control group,OD value and the expression of IL-8,TNF-α,PCNA,PI3K,p-AKT in Girdin-siRNA group and cisplatin group were significantly decreased,the apoptosis rate and the expression of Bax and Caspase-3 were significantly increased(P<0.05);OD value and the expression of IL-8,TNF-α,PCNA,PI3K,p-AKT in Girdin-siRNA+cisplatin group were significantly lower than Girdin-siRNA group and cisplatin group,the apoptosis rate and the expression of Caspase-3 and Bax were significantly higher than Girdin-siRNA group and cisplatin group(P<0.05).Conclusion:Inhibiting of Girdin expression in lung cancer cells can reduce the proliferation of cancer cells,induce apoptosis and improve immunity to enhance the sensitivity of cisplatin chemotherapy.The mechanism of cell proliferation and apoptosis may be related to the downregulation of PI3K/AKT signaling pathway.