5-Aza-dC对硫酸铍诱导A549细胞中TβRⅡDNA甲基化的作用  

Effect of 5-aza-dC on TβRⅡDNA methylation induced by beryllium sulfate in A549 cells

作　　者：刘科良 孙林清 王耀峰 李思睿 徐海明 刘志宏 LIU Ke-liang;SUN Lin-qing;WANG Yao-feng;LI Si-rui;XUHai-ming;LIU Zhi-hong(School of Health Management,SanQuan College of Xinxiang Medical University,Xinxiang Henan 453003,China;School of Public Health and Management,Ningxia Medical University,Yinchuan Ningxia 750000,China;Key Laboratory of Environmental Factors and Chronic Disease Control,Yinchuan Ningxia 750000,China)

机构地区：[1]新乡医学院三全学院健康管理学院,河南新乡453003 [2]宁夏医科大学公共卫生与管理学院,宁夏银川750000 [3]宁夏环境因素与慢性病控制重点实验室,宁夏银川750000

出　　处：《毒理学杂志》2022年第6期485-489,共5页Journal of Toxicology

基　　金：国家自然科学基金(30020802);教育部“春晖计划”项目(Z2016052)。

摘　　要：目的探讨5-氮杂-2’-脱氧胞苷(5-Aza-2’-deoxycytidine,5-Aza-dC)对BeSO_(4)诱导人肺腺癌A549细胞中转化生长因子-βⅡ型受体(TβRⅡ)DNA甲基化的作用。方法建立BeSO_(4)染毒的A549细胞培养实验模型,采用CCK-8法检测细胞增殖活性,荧光定量聚合酶链反应(Quantitative Real-time PCR)检测TβRⅡ、α-平滑肌肌动蛋白(α-SMA)、胶原蛋白Ⅲ(COL-Ⅲ)和胶原蛋白Ⅰ(COL-Ⅰ)的mRNA表达;用5-Aza-dC进行干预,采用巢式降落式甲基化特异性PCR法检测TβRⅡDNA甲基化水平,用蛋白质印迹法(Western blot)检测TβRⅡ蛋白相对表达水平。结果根据细胞增殖结果建立低、中、高剂量组(终浓度分别为1、10和100μmol/L);BeSO_(4)可以诱导细胞形态变化,从椭圆形紧密连接的上皮细胞到纺锤形松散连接的间质细胞,并且随着暴露时间的延长,形态变化更加明显,BeSO_(4)在不同时间内诱导的细胞存活率随暴露时间延长而降低,最终选用细胞存活相对较多且低、中、高剂量组差异均有统计学意义的48 h作为干预时间;PCR结果显示:与对照组相比,BeSO_(4)各剂量组α-SMA、COL-Ⅲ和COL-Ⅰ的mRNA表达水平升高,这提示A549细胞胶原过量形成;与对照组相比,BeSO_(4)中、高剂量组的TβRⅡDNA甲基化水平分别增加77.52%和96.71%(P<0.05),5-Aza-dC干预组TβRⅡDNA甲基化水平较中剂量组降低28.60%(P<0.05);与对照组相比,BeSO_(4)低、中剂量组TβRⅡmRNA表达水平升高,中、高剂量组TβRⅡ蛋白相对表达水平分别升高117.49%、203.08%(P<0.05);5-Aza-dC干预后,TβRⅡ蛋白的表达水平较中剂量组降低15.22%,差异有统计学意义(P<0.05)。结论5-Aza-dC干预可导致BeSO_(4)诱导的A549细胞TβRⅡDNA甲基化程度降低。Objective To investigate the effect of 5-Aza-2′-deoxycytidine(5-Aza-dC)on effect of DNA methylation of BeSO_(4)-induced transforming growth factor-βtypeⅡreceptor in A549 cells.Methods The experimental model of A549 cells exposed to BeSO_(4)was established.The cell proliferation activity was detected by cell counting kit-8(CCK-8),and TβRⅡ,α-SMA,COL-Ⅲand COL-Ⅰwere detected by Quantitative Real-time PCR,5-aza-dC was used for intervention,and nested landing methylation specific PCR was used to detect TβRⅡDNA methylation level,the relative protein expression level was detected by Western blot.Results According to the result of cell proliferation,low,medium and high dose groups were established,BeSO_(4)can induce cell morphological changes,from oval tightly connected epithelial cells to spindle loosely connected stromal cells,and the morphological changes are more obvious with the extension of exposure time.The cell survival rate induced by BeSO_(4)at different times was decreased with the extension of exposure time.Finally,48 h with relatively more cell survival and statistically significant difference among low,medium and high dose groups was selected as the intervention time;Compared with the control group,PCR result showed that the mRNA expression levels ofα-SMA,COL-Ⅲand COL-Ⅰin BeSO_(4)groups were increased,suggesting the excessive formation of collagen in A549 cells;Compared with the control group,the DNA methylation levels of TβRⅡin BeSO_(4)medium and high dose groups were increased by 77.52%and 96.71%respectively(P<0.05),the DNA methylation level of TβRⅡwas 28.60%lower than that in the medium dose group(P<0.05);Compared with the control group,the expression level of TβRⅡmRNA increased in BeSO_(4)low and medium dose groups,and the relative expression levels of TβRⅡprotein in medium and high dose groups increased by 117.49%and 203.08%respectively(P<0.05);After 5-aza-dC intervention,the expression level of TβRⅡprotein was 15.22%lower than that in the medium dose group(P<0.05).Co

关 键 词：转化生长因子-βⅡ型受体 DNA甲基化 5-AZA-DC 

分 类 号：R114[医药卫生—卫生毒理学] R734.2[医药卫生—公共卫生与预防医学] R99