p38M APK基因对PM_(2.5)染毒HK-2细胞部分癌基因和凋亡相关基因表达的影响  

Effects of p38MAPK gene on PM_(2.5)-induced expression of oncogenes and apoptosis genes in HK-2 cells

作　　者：李柏茹 秦双建 李闰冰 蔡颖[2,3] 郑凯[2,3] 曾明 肖芳[1] 徐新云[2] LI Boru;QIN Shuangjian;LI Runbing;CAI Ying;ZHENG Kai;ZENG Ming;XIAO Fang;XU Xinyun(Xiangya School of Public Health,Central South University,Changsha 410078,Hunan;Shenzhen Center for Disease Control and Prevention,Shenzhen 518055,Guangdong;School of Public Health,University of South China,Hengyang 421001,Hunan,China)

机构地区：[1]中南大学湘雅公共卫生学院,湖南长沙410078 [2]深圳市疾病预防控制中心,广东深圳518055 [3]南华大学公共卫生学院,湖南衡阳421001

出　　处：《癌变．畸变．突变》2021年第2期129-135,142,共8页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：深圳市科技研发项目(JCYJ20170413101713324,JCYJ20190807102205480)。

摘　　要：目的:探讨p38MAPK基因对PM_(2.5)染毒人肾上皮细胞(HK-2)部分癌基因和凋亡相关基因表达的影响。方法:根据GenBank提供的p38MAPK mRNA序列,设计合成干扰序列,将重组慢病毒载体转染HK-2细胞,构建p38MAPK基因沉默细胞株。分别采用实时荧光定量PCR(qPCR)和Western blot法检测p38MAPK mRNA和蛋白的表达水平鉴定沉默效果。用50μg/mL的PM_(2.5)混悬液分别染毒正常HK-2细胞和p38MAPK基因沉默细胞24 h,利用荧光定量PCR和Western blot检测癌基因c-myc,c-fos、p53和凋亡相关基因Caspase-8、Caspase-9、Bcl-2的mRNA和蛋白的相对表达水平。结果:qPCR和Western blot检测结果显示,与正常HK2细胞比较,p38MAPK基因沉默细胞中p38MAPK mRNA的表达下降了58.50%,p38MAPK蛋白表达水平下降了51.33%(P<0.01)。PM_(2.5)混悬液对HK-2细胞和p38MAPK基因沉默HK-2细胞染毒后,qPCR检测结果显示,与正常HK-2细胞未染毒组比较,正常HK-2细胞PM_(2.5)染毒组中c-myc、c-fos、Caspase-8和Casepase-9基因表达分别升高39.89%、15.12%、19.47%和15.45%,p53和Bcl-2基因表达分别下降21.54%和31.77%,差异均具有统计学意义(P<0.05);与正常HK-2细胞PM_(2.5)染毒组比较,p38MAPK基因沉默HK-2细胞PM_(2.5)染毒组中c-myc、c-fos、p53、Caspase-8和Caspase-9 mRNA表达分别下降了84.55%、63.55%、34.49%、37.19%和54.97%,差异均具有统计学意义(P<0.05)。Western blot检测结果显示,与正常HK-2细胞未染毒组比较,正常HK-2细胞PM_(2.5)染毒组中的c-myc和Caspase-8蛋白表达增加,Bcl-2蛋白表达减少;与正常HK-2细胞PM_(2.5)染毒组比较,p38MAPK基因沉默HK-2细胞PM_(2.5)染毒组中的c-myc、Caspase-8和Bcl-2蛋白表达减少(均为P<0.05)。结论:成功构建了p38MAPK基因沉默细胞株,PM_(2.5)可引起HK-2细胞癌基因和凋亡相关基因表达水平升高,p38MAPK基因可能参与PM_(2.5)对HK-2细胞的毒性作用过程。OBJECTIVE:To investigate effects of p38MAPK gene on expression of oncogenes and apoptosis-related genes in human kidney epithelial cells(HK-2 cells)which had been exposed to PM_(2.5).METHODS:According to the mRNA sequence of p38MAPK gene provided by GenBank,interference sequences were designed and synthesized,and a recombinant lentiviral vector was constructed and was transfected into HK-2 cells to construct p38MAPK gene-silenced cells.Real-time fluorescent quantitative PCR(qPCR)and western blot were used to identifyeffects from p38MAPK gene silencing.HK-2 cells and p38MAPK gene-silenced cells were treated with 50μg/mL PM_(2.5) suspension for 24 h,and qPCR and western blot were used to detect their expression of oncogenes(c-myc,c-fos and p53)and apoptosis-related genes(Caspase-8,Caspase-9 and Bcl-2).RESULTS:p38MAPK mRNA expression levels in p38MAPK-silenced cells were inhibited by 58.50%and p38MAPK protein expression levels by 51.33%when compared with the normal HK-2 cells(P<0.01).These results indicate that the p38MAPK silenced cells were successfully constructed.The qPCR results showed that when compared with the HK-2 cells in the control group,the PM_(2.5)-exposed cells indicated the mRNA of c-myc,c-fos,Caspase-8 and Casepase-9 in expressions were significantly increased by 39.89%,15.12%,19.47%and 15.45%,respectively,and p53 and Bcl-2 mRNA expressions were significantly decreased by 21.54%and 31.77%respectively(P<0.05).When compared with the PM_(2.5) exposed HK-2 cells,the PM_(2.5)-exposed p38MAPK-silenced cells showed that the mRNA of c-myc,c-fos,p53,Caspase-8 and Caspase-9 expressions were significantly decreased by 84.55%、63.55%、34.49%、37.19%and 54.97%respectively,(P<0.05).At the protein level,when compared with the HK-2 cells in the control group,the PM_(2.5)-exposed cells showed that the protein levels of c-myc,and Caspase-8 were increased and that of Bcl-2 were decreased.When compared with the PM_(2.5)-exposed HK-2 cells,the PM_(2.5)-exposed p38MAPK-silenced cells showed that the mRNA levels of c-

关 键 词：p38MAPK 大气细颗粒物 人肾上皮细胞 基因沉默 癌基因 凋亡相关基因 

分 类 号：R122.7[医药卫生—环境卫生学]