K-ras基因沉默对PM_(2.5)染毒HBE细胞癌基因表达的影响  

Effects of K-ras gene silence on the expression of oncogenes in HBE cells treated with PM_(2.5)

作　　者：李闰冰 蒲桔宁 李柏茹 蔡颖[1,2] 张朝晖[1] 徐新云[2] Li Runbing;Pu Juning;Li Boru;Cai Ying;Zhang Zhaohui;Xu Xinyun(School of Public Health,University of South China,Hengyang 421001,China;Institute of Environment and Health,Shenzhen Center for Disease Control and Prevention,Shenzhen 518055,China;Xiangya School of Public Health,Central South University,Changsha 410078,China)

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

出　　处：《中华劳动卫生职业病杂志》2021年第11期825-830,共6页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：深圳市科技研发基础研究项目(JCYJ20190807102205480);深圳市医疗卫生三名工程(SZSM201611068);深圳市医学重点学科建设经费(SZXK067)。

摘　　要：目的探讨K-ras基因对PM_(2.5)染毒人支气管上皮(HBE)细胞部分癌基因和抑癌基因表达的影响。方法于2019年9月,根据K-ras基因mRNA序列,设计合成干扰序列,转染HBE细胞构建K-ras基因沉默细胞。用10、50μg/ml PM_(2.5)混悬液及10μmol/L Cr^(6+)分别染毒HBE细胞和K-ras基因沉默细胞,实时荧光定量PCR检测c-myc、c-fos、N-ras、cyclin-D1、p16、p53基因的mRNA表达水平,Western blot检测c-myc和p53蛋白表达水平。结果K-ras基因沉默细胞中K-ras基因mRNA表达水平下降(80.5%±3.6%),K-ras蛋白表达水平下降(58.9%±4.7%)(P<0.01)。各浓度PM_(2.5)染毒HBE细胞组、K-ras沉默细胞组及10μmol/L Cr^(6+)染毒HBE细胞组、K-ras沉默细胞组c-myc、c-fos、N-ras、cyclin-D1基因mRNA表达水平均高于未染毒相应细胞对照组,p16和p53基因mRNA表达水平低于未染毒相应细胞对照组(P<0.01);10μg/ml PM_(2.5)染毒K-ras沉默细胞组c-myc、c-fos、p16基因mRNA表达水平低于10μg/ml PM_(2.5)染毒HBE细胞组,p53基因mRNA表达水平高于10μg/ml PM_(2.5)染毒HBE细胞组(P<0.01);50μg/ml PM_(2.5)染毒K-ras沉默细胞组c-fos、N-ras、cyclin-D1、p16和p53基因mRNA表达水平均低于50μg/ml PM_(2.5)染毒HBE细胞组(P<0.01)。50μg/ml PM_(2.5)染毒HBE细胞组c-myc蛋白表达水平高于未染毒HBE细胞组,p53蛋白表达水平低于未染毒HBE细胞组(P<0.05);50μg/ml PM_(2.5)染毒K-ras沉默细胞组c-myc蛋白表达水平高于未染毒K-ras沉默细胞组(P<0.05)。结论PM_(2.5)可引起HBE细胞癌基因表达升高,K-ras基因沉默能抑制PM_(2.5)诱导HBE细胞癌基因的表达。Objective To explore the effects of K-ras gene on the expressions of oncogenes and cancer suppressor genes in human bronchial epithelial(HBE)cells which were exposed to PM_(2.5).Methods According to the mRNA sequence of K-ras gene provided by GenBank in September 2019,interference sequences were designed and synthesized,and the recombinant lentiviral vector was transfected into HBE cell to construct the K-ras gene-silenced cells.HBE cells and K-ras gene-silenced cells were exposed to 10μg/ml,50μg/ml PM_(2.5) suspension and 10μmol/L Cr^(6+).Real-time fluorescent quantitative PCR was used to detect the mRNA expression levels of c-myc,c-fos,N-ras,cyclin-D1,p16 and p53 genes,the expression levels of p53 and c-myc proteins were detected by Western blot.Results In K-ras silenced cell group,K-ras mRNA expression level decreased(80.5%±3.6%)and K-ras protein level decreased(58.9%±4.7%)when compared with the control group(P<0.01).Compared with the correspoding cell control group without exposure,the mRNA expression levels of c-myc,c-fos,N-ras and cyclin-D1 genes in HBE cell group exposed to different concentrations of PM_(2.5),K-ras silenced cell group exposed to different concentrations of PM_(2.5),HBE cell group exposed to 10μmol/L Cr^(6+)and K-ras silenced cell group exposed to 10μmol/L Cr^(6+)were increased,the mRNA expressions of p16 and p53 genes were decreased(P<0.01).Compared with HBE cell group exposed to 10μg/ml PM_(2.5),the mRNA expressions of c-myc,c-fos and p16 genes in K-ras silenced cells exposed to 10μg/ml PM_(2.5) were decreased,and the p53 mRNA level was increased(P<0.01).Compared with HBE cell group exposed to 50μg/ml PM_(2.5),the mRNA expression levels of c-fos,N-ras,cyclin-D1,p16 and p53 genes in K-ras silenced cell group exposed to 50μg/ml PM_(2.5) were decreased(P<0.01).Compared with the HBE cell group without exposure,c-myc protein increased and p53 protein decreased in HBE cells exposed to 50μg/ml PM_(2.5)(P<0.05).Compared with the K-ras silenced cell group without exposure,c-myc protei

关 键 词：基因沉默 K-RAS 细颗粒物 人支气管上皮细胞 癌基因 

分 类 号：R114[医药卫生—卫生毒理学]