癌变·畸变·突变 ›› 2020, Vol. 32 ›› Issue (4): 298-303,308.doi: 10.3969/j.issn.1004-616x.2020.04.009

• 论著 • 上一篇    下一篇

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

蔡颖1,2, 郑凯1,2, 秦双建2,3, 李柏茹2,3, 余淑苑2, 刘宁2, 季佳佳2, 张朝晖1, 徐新云2   

  1. 1. 南华大学公共卫生学院, 湖南 衡阳 421001;
    2. 深圳市疾病预防控制中心, 广东 深圳 518055;
    3. 中南大学湘雅公共卫生学院, 湖南 长沙 410078
  • 收稿日期:2020-04-21 修回日期:2020-07-01 出版日期:2020-07-31 发布日期:2020-08-01
  • 通讯作者: 张朝晖,E-mail:zhaohuizzh@126.com;徐新云,E-mail:xyxu2008@163.com E-mail:zhaohuizzh@126.com;xyxu2008@163.com
  • 作者简介:蔡颖,E-mail:1479288556@qq.com。
  • 基金资助:
    深圳市科技研发基础研究项目（JCYJ20170413101713324）

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

CAI Ying1,2, ZHENG Kai1,2, QIN Shuangjian2,3, LI Boru2,3, YU Shuyuan2, LIU Ning2, JI Jiajia2, ZHANG Zhaohui1, XU Xinyun2   

  1. 1. School of Public Health, University of South China, Hengyang 421001, Hunan;
    2. Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, Guangdong;
    3. Xiangya School of Public Health, Central South University, Changsha 410078, Hunan, China
  • Received:2020-04-21 Revised:2020-07-01 Online:2020-07-31 Published:2020-08-01

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摘要： 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50 μg/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 mRNA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。

关键词: c-fos, 大气细颗粒物, 人支气管上皮细胞, 基因沉默, 癌基因, 凋亡相关基因

Abstract: 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 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 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 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 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.

Key words: c-fos, PM2.5, human bronchial epithelial cells, gene silence, oncogene, apoptosis gene

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