沉默c-fos基因对PM2.5染毒人支气管上皮细胞癌基因和凋亡相关基因表达的影响

Silencing of c-fos gene on expression of oncogenes and apoptosis genes in HBE cells exposed to PM2.5

目的:探讨沉默c-fos基因对PM2.5染毒人支气管上皮细胞(HBE细胞)癌基因和凋亡相关基因表达的影响。方法:采用RNA干扰技术,根据GenBank提供的c-fos mRNA序列,设计合成shRNA干扰序列,将重组慢病毒载体转染HBE细胞,构建c-fos基因沉默细胞株。采用实时荧光定量PCR(qPCR)和Western blot对c-fos基因沉默效果进行鉴定。以HBE细胞、c-fos基因沉默细胞为实验对象,用浓度为50μg/mL的PM2.5混悬液染毒24 h,同时设立阴性对照组,qPCR和Western blot分别检测部分癌基因c-myc、k-ras、c-fos、p53和凋亡相关基因Caspase-3、Caspase-8在mRNA及蛋白质表达水平的变化。结果:转染c-fos-shRNA慢病毒的HBE细胞中,经qPCR和Western blot检测发现,与对照组HBE细胞相比,c-fos m RNA表达水平下降70.1%,c-fos蛋白表达水平下降53.7%。PM2.5染毒HBE细胞后,与未染毒的对照组比较,c-myc和k-ras mRNA表达水平分别升高30.46%、27.17%,差异均有统计学意义(P<0.05),c-fos、Caspase-3、Caspase-8 mRNA表达水平分别升高50.78%、71.91%、74.16%,p53 mRNA表达水平下降12.27%,差异均有统计学意义(P<0.01)。PM2.5染毒c-fos基因沉默细胞后,与未染毒组比较,c-myc、k-ras mRNA表达水平分别下降13.08%、5.39%,Caspase-3、Caspase-8 mRNA表达水平分别下降25.03%、21.37%(P<0.05),p53 mRNA下降53.39%(P<0.01)。此外,c-fos基因沉默细胞染毒后,与HBE正常细胞染毒后比较,c-myc、k-ras、p53、c-fos、Caspase-3和Caspase-8的mRNA表达水平均有所下降(P<0.05);仅c-myc蛋白表达下降(P<0.01)。结论:成功构建c-fos基因沉默细胞株;PM2.5可引起HBE细胞癌基因和凋亡相关基因表达水平明显升高,c-fos基因沉默在一定程度上能抑制PM2.5对癌基因和凋亡相关基因的激活作用。

OBJECTIVE:To explore the effect of c-fos gene silencing on expression of oncogenes and apoptosis-related genes in human bronchial epithelial(HBE)cells which were exposed to PM2.5.METHODS:According to the c-fos gene mRNA sequence provided by GenBank,interfering sequences were designed and synthesized,and the recombinant lentiviral vector was transfected into HBE cells.Real-time quantitative PCR(qPCR)and Western blot were used to identify the silencing of c-fos gene.HBE cells and c-fos gene-silenced cells were exposed to PM2.5 suspension with a concentration of 50μg/mL for 24 h,and a non-exposed control group was established.The mRNA levels and protein levels of oncogenes including c-myc,k-ras,c-fos,p53 and apoptosis-related genes including Caspase-3,Caspase-8 were detected by qPCR and Western blot.RESULTS:In HBE cells transfected with c-fos-shRNA lentivirus,qPCR and Western blot analyses revealed that expression of c-fos gene mRNA was 70.1%lower,and expression of c-fos protein was 53.7%lower than that of control group HBE cells.After HBE cells were treated with PM2.5,mRNA expression of c-myc,k-ras,c-fos,Caspase-3,and Caspase-8 were increased by 30.46%,27.17%,and 50.78%,71.91%,74.16%,respectively,compared with the untreated HBE cells,p53 mRNA expression level decreased by 12.27%(P<0.05).After the c-fos gene silenced cells were treated with PM2.5,mRNA expression of c-myc,k-ras,Caspase-3,and Caspase-8 decreased by 13.08%,5.39%,25.03%,21.37%,respectively,and p53 mRNA decreased by 53.39%,compared with the untreated group.In comparison with normal HBE cells,mRNA expression of cmyc,k-ras,p53,c-fos,Caspase-3,and Caspase-8 were significantly reduced(P<0.05).Furthermore,protein level of c-myc also showed a decreasing trend after c-fos gene-silenced cells were exposed to PM2.5,compared with that of normal HBE cells.CONCLUSION:c-fos gene-silenced cells were successfully constructed.PM2.5 promoted expression of oncogenes and apoptotic genes in HBE cells.On the other hand,c-fos gene silencing inhibited the expression of oncogenes and apoptotic genes after the PM2.5 treatment in HBE cells.