c-myc基因沉默对PM2.5染毒L02肝细胞癌基因和凋亡基因表达的影响  

Effect of c-myc gene silence on the expression of oncogenes and apoptotic genes in hepatocytes treated with PM2.5

作　　者：秦双建 王冰玉 李柏茹 郑凯 蔡颖[2,3] 李闰冰[3] 曾明 肖芳[1] 徐新云[2] Qin Shuangjian;Wang Bingyu;Li Boru;Zheng Kai;Cai Ying;Li Runbing;Zeng Ming;Xiao Fang;Xu Xinyun(Xiangya School of Public Health,Central South University,Changsha 410078,China;Institute of Environment and Health,Shenzhen Center for Disease Control and Prevention,Shenzhen 518055,China;School of public health,University of South China,Hengyang 421001,China)

机构地区：[1]中南大学湘雅公共卫生学院,长沙410078 [2]深圳市疾病预防控制中心环境与健康所,518055 [3]南华大学公共卫生学院,衡阳421001

出　　处：《中华劳动卫生职业病杂志》2020年第9期657-663,共7页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：深圳市科技研发基础研究项目(JCYJ20170413101713324)。

摘　　要：目的构建c-myc基因沉默肝细胞株,研究c-myc基因沉默对PM2.5染毒L02肝细胞凋亡基因和癌基因表达的影响。方法根据GenBank提供的c-myc基因mRNA序列,设计合成3条干扰序列,将重组慢病毒载体转染L02肝细胞。利用实时荧光定量PCR(Q-PCR)和蛋白免疫印迹法(Western blotting)对c-myc基因沉默效果进行鉴定。用L02肝细胞、c-myc基因沉默细胞作为试验对象,用浓度为50μg/ml的PM2.5水溶物和10μmol/L阳性对照Cr^6+染毒24 h即PM2.5组、Cr^6+组,同时设立空白对照组;荧光定量PCR检测癌基因(c-myc、c-fos、k-ras、p53)和凋亡基因[半胱氨酸蛋白酶(Caspase)-3、Caspase-8、Caspase-9]的mRNA相对表达水平;Western blotting检测癌基因和凋亡基因的蛋白质表达水平。结果与正常L02肝细胞比较,c-myc基因沉默细胞的c-myc基因表达明显受到抑制,其mRNA表达量下降81%。c-myc蛋白表达下降70%(P<0.01)。与正常L02肝细胞比较,PM2.5水溶物染毒组c-myc、c-fos、k-ras表达水平分别下降84.1%、45.4%、54.6%,p53表达升高192.9%、Caspase-3、Caspase-8、Caspase-9表达分别下降24.4%、36.1%、60.9%(P<0.05)。Cr^6+阳性对照组c-myc、c-fos、k-ras表达分别下降72.1%、82.2%、54.0%,p53表达升高250.0%,Caspase-3、Caspase-8、Caspase-9表达分别下降34.6%、36.0%、68.9%(P<0.05)。与正常L02肝细胞比较,PM2.5染毒组c-myc和c-fos蛋白表达量升高,p53蛋白表达量下降,Caspase-3、Caspase-8、Caspase-9蛋白表达升高(P<0.05);与c-myc基因沉默细胞比较,PM2.5染毒组c-myc和c-fos蛋白表达量下降,p53蛋白表达量上升(P<0.05)。结论PM2.5可引起L02肝细胞癌基因和凋亡基因表达水平明显升高,c-myc基因沉默在一定程度上能抑制PM2.5对凋亡基因和癌基因的激活作用。Objective To construct the c-myc gene silenced hepatocytes,study the effect of c-myc gene silence on expression of oncogenes and apoptosis genes in hepatocytes treated with PM2.5.Methods According to the c-myc gene mRNA sequence provided by GenBank,three interfering sequences were designed and synthesized,the recombinant lentiviral vector was transfected into L02 hepatocytes.The real-time quantitative PCR and western blotting were used to identify the effect of c-myc gene silencing.L02 cells and c-myc gene silenced cells were used as experimental subjects.The normal L02 cells and c-myc silenced cells were treated with 50μg/ml PM2.5 water soluble solution,10μM positive control Cr^6+and a blank control,the treatment period was 24 h.The mRNA levels of oncogenes(c-myc,c-fos,k-ras,p53)and apoptotic genes(Caspase-3,Caspase-8,Caspase-9)were detected by real-time PCR.The protein levels of oncogenes and apoptotic genes were detected by western blotting.Results The mRNA level and protein level of c-myc decreased by 81%and 70%in c-myc silenced cells when compared with the normal L02 hepatocytes,the above results indicate that c-myc gene silenced cells were successfully constructed.After c-myc silenced cells were treated with PM2.5 water soluble solution,The mRNA levels of c-myc,c-fos,and k-ras decreased by 84.1%,45.4%,and 54.6%(P<0.05),p53 increased by 192.9%(P<0.05),and the expression of Caspase-3,Caspase-8,and Caspase-9 decreased by 24.4%,36.1%,60.9%(P<0.05).In the Cr^6+positive control group,the expression of c-myc,c-fos,and k-ras decreased by 72.1%,82.2%,and 54.0%(P<0.05),p53 increased by 250.0%(P<0.05),the expression of Caspase-3,Caspase-8,and Caspase-9 decreased by 34.6%,36.0%,68.9%(P<0.05),respectively,when compared with the normal L02 hepatocytes(P<0.05).Western blotting results showed that the protein levels of c-myc and c-fos increased,p53 decreased after PM2.5 exposure;the protein levels of Caspase-3,Caspase-8,Caspase-9 increased after PM2.5 exposure(P<0.05).When in comparison with the c-myc silenced group,the

关 键 词：颗粒物 细颗粒物 肝细胞 基因沉默 癌基因 凋亡基因 

分 类 号：R735.7[医药卫生—肿瘤]