砷及其代谢物对BEAS-2B细胞p53基因表达影响  

Effects of arsenic and its metabolites on p53 gene expression in BEAS-2B cells

作　　者：刘娜 江金鋆 马和 赵瑞欢 何越峰 文卫华[1] LIU Na;JIANG Jinjun;MA He;ZHAO Ruihuan;HE Yuefeng;WEN Weihua(Yunnan Center for Disease Control and Prevention and Yunnan Provincial Key Laboratory of Public Health and Biosafety,Kunming,Yunnan 650022,China;College of Public Health,Kunming Medical University,Kunming,Yunnan 650500,China)

机构地区：[1]云南省疾病预防控制中心暨云南省公共卫生与生物安全重点实验室,云南昆明650022 [2]昆明医科大学公共卫生学院,云南昆明650500

出　　处：《环境与职业医学》2024年第4期431-436,共6页Journal of Environmental and Occupational Medicine

基　　金：国家自然科学基金地区科学基金项目(82060591);云南省万人计划-名医专项(YNWR-MY-2018-012);云南省医学领军人才培养计划(L-2018016)。

摘　　要：[背景]砷是人类致癌物。砷及其代谢物会影响p53表达,但砷及其代谢物处理人正常肺上皮细胞系(BEAS-2B)后,p53磷酸化及泛素化水平的变化尚不清楚。[目的]探讨砷及其代谢物一甲基胂酸(MMA)和二甲基胂酸(DMA)对BEAS-2B细胞中抑癌基因p53表达的影响。用不同浓度亚砷酸钠(NaAsO2)染毒细胞,利用CCK-8试剂检测细胞活力,确定NaAsO2染毒BEAS-2B细胞的剂量和时间。基于细胞活力结果,将细胞分为两组,即不同浓度亚砷酸钠组和砷甲基化代谢产物组,不同浓度亚砷酸钠组染毒剂量分别为0、2、4、6μmol·L^(-1)NaAsO2;砷甲基化代谢产物组包括0μmol·L^(-1)NaAsO2(对照组)、6μmol·L^(-1)MMA组、6μmol·L^(-1)DMA组和6μmol·L^(-1)NaAsO2组。48 h收集细胞,提取总蛋白和总RNA,采用实时荧光定量聚合酶链式反应(qRT-PCR)检测p53 mRNA相对表达水平,采用免疫共沉淀和蛋白质免疫印迹法检测p53泛素化水平,通过蛋白免疫印迹法检测p53蛋白、p53 Ser9和Ser15位点磷酸化蛋白的相对表达水平。[结果]在BEAS-2B细胞中,与对照组相比,不同浓度NaAsO2染毒后,细胞存活率均降低(P<0.05),并且NaAsO2浓度在6μmol·L^(-1)时细胞存活率约为50%。与对照组相比,不同浓度的NaAsO2(2、4、6μmol·L^(-1))处理后p53 mRNA相对表达水平呈逐渐降低趋势,差异具有统计学意义(P<0.05);不同浓度的NaAsO2诱导p53蛋白、Ser9位点磷酸化蛋白相对表达水平均逐渐降低(P<0.05);不同浓度的NaAsO2诱导p53 Ser15位点磷酸化蛋白相对表达水平均呈逐渐降低趋势,但仅6μmol·L^(-1)NaAsO2组低于对照组且差异具有统计学意义(P<0.05)。与对照组相比,MMA组和DMA组p53 mRNA、p53蛋白、Ser9和Ser15位点磷酸化蛋白相对表达水平无明显影响。与对照组相比,NaAsO2染毒后,p53泛素化表达水平明显下降,K48位点泛素化表达水平下降。[结论]砷引起BEAS-2B细胞p53蛋白表达降低主要由于其磷酸化途径被抑制和mRNA表�[Background]Arsenic is a human carcinogen.Arsenic and its metabolites affect the expression of p53,but whether there are any changes of p53 phosphorylation and ubiquitination levels in human bronchial epithelium cells(BEAS-2B)are not clear after exposure to arsenic and its metabolites.[Objective]To study the effects of arsenic and its metabolites monomethylarsic acid(MMA)and dimethylarsinic acid(DMA)on the expression of tumor suppressor gene p53 in BEAS-2B cells.[Methods]Different concentrations of sodium arsenite(NaAsO_(2))were used to infect BEAS-2B cells,and the cell viability was detected with CCK-8 reagent to determine the dose and time of NaAsO_(2) used for the following study.Based on the results of cell viability,the cells were divided into two panels:a sodium arsenide panel and an arsenic methylation metabolite penal.The doses of sodium arsenite were 0,2,4,and 6μmol·L^(−1) NaAsO_(2);the arsenic methylation metabolite panel consisted of 0μmol·L^(−1) NaAsO_(2) group(control),6μmol·L^(−1) MMA group,6μmol·L^(−1) DMA group,and 6μmol·L^(−1) NaAsO_(2) group.The cells were collected after 48 h treatment,and the total protein and total RNA were extracted.The relative levels of p53 mRNA expression were determined by quantitative real-time polymerase chain reaction(qRT-PCR),the relative expression levels of p53 protein,p53 Ser9 and Ser15 phosphorylated proteins were determined by Western blot,and the level of p53 ubiquitination was detected by co-immunoprecipitation(CO-IP).[Results]Compared with the control group,the cell viability rates in all BEAS-2B cells treated by NaAsO_(2) were significantly reduced(P<0.05),and the 50%cell viability was observed at 6μmol·L^(−1).Compared with the control group,the relative expression level of p53 mRNA gradually decreased after NaAsO_(2)(2,4,6μmol·L^(−1))treatment(P<0.05),the relative expression levels of p53 protein and Ser9 phosphorylated protein induced by NaAsO_(2) also decreased gradually(P<0.05),and the relative expression level of p53 Ser15 ph

关 键 词：砷 P53 一甲基胂酸 二甲基胂酸 泛素化 

分 类 号：R11[医药卫生—公共卫生与预防医学]