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

目的： 探讨沉默c-fos基因对PM_2.5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50 μg/mL的PM_2.5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 mRNA表达水平下降70.1%，c-fos蛋白表达水平下降53.7%。PM_2.5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）。PM_2.5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；PM_2.5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PM_2.5对癌基因和凋亡相关基因的激活作用。

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

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 PM_2.5. 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 PM_2.5 suspension with a concentration of 50 μg/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 PM_2.5, 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 PM_2.5, 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 c-myc,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 PM_2.5,compared with that of normal HBE cells. CONCLUSION: c-fos gene-silenced cells were successfully constructed. PM_2.5 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 PM_2.5 treatment in HBE cells.