亚砷酸钠致人肝星状细胞活化中铁死亡相关基因SLC7A11和CDKN1A的DNA甲基化及表达变化  

DNA methylation and expression changes of ferroptosis related genes SLC7A11 and CDKN1A in human hepatic stellate cell activation induced by sodium arsenite

作　　者：赵丽君 丁关鑫 黄菲 迪丽娜尔·亚尔麦麦提 吴顺华 ZHAO Lijun;DING Guanxin;HUANG Fei;Dilina’er YA’ERMAIMAITI;WU Shunhua(Department of Occupational and Environmental Health,School of Public Health,Xinjiang Medical University,Urumqi,Xinjiang 830011,China;Department of Epidemiology and Health Statistics,School of Public Health,Xinjiang Medical University,Urumqi,Xinjiang 830011,China)

机构地区：[1]新疆医科大学公共卫生学院劳动卫生与环境卫生学教研室,新疆乌鲁木齐830011 [2]新疆医科大学公共卫生学院流行病与卫生统计学教研室,新疆乌鲁木齐830011

出　　处：《环境与职业医学》2023年第12期1403-1410,共8页Journal of Environmental and Occupational Medicine

基　　金：国家自然科学基金项目(82160650)。

摘　　要：[背景]无机砷在肝脏内蓄积易导致肝纤维化。溶质载体家族7成员11(SLC7A11)和细胞周期蛋白依赖性激酶抑制因子1A(CDKN1A)是铁死亡相关基因,在肝脏及纤维化疾病中异常表达,但其在无机砷致肝纤维化的作用尚未见报道。[目的]探究不同剂量的亚砷酸钠对人肝星状细胞(LX-2)迁移能力的影响及亚砷酸钠致LX-2细胞活化中的SLC7A11、CDKN1A DNA甲基化和表达的变化。[方法]体外培养LX-2细胞株,用不同剂量的亚砷酸钠处理LX-2细胞24 h,实验分为对照组(0μmol·L^(−1))、低剂量组(5μmol·L^(−1))、中剂量组(10μmol·L^(−1))、高剂量组(15μmol·L^(−1))。倒置显微镜观察各组细胞形态变化;划痕实验检测细胞迁移运动情况;透射电镜观察对照组和高剂量组线粒体超微结构;FerroOrange荧光探针检测细胞Fe^(2+);运用Illumina 850K甲基化芯片对差异甲基化位点进行筛查;实时荧光定量PCR和Western blot检测铁死亡指标(SLC7A11、CDKN1A)及人肝星状细胞活化指标[转化生长因子β1(TGF-β1)、Ⅲ型胶原(CollagenⅢ)]。[结果]随着染毒剂量的升高,细胞触角回缩逐渐变为圆形,且细胞划痕愈合情况逐渐变差。透射电镜观察到高剂量组部分细胞线粒体膜完整性破坏、线粒体嵴断裂。激光共聚焦显微镜观察到Fe^(2+)荧光强度随着染毒剂量的升高而逐渐升高。甲基化芯片结果显示,与对照组相比,高剂量组SLC7A11在启动子区cg22659014位点高甲基化,CDKN1A在启动子区cg17964532位点高甲基化。随染毒剂量的增加,SLC7A11 mRNA及蛋白表达水平降低(P<0.001);各组间CDKN1A mRNA水平无明显变化(P>0.05),低、中、高剂量组CDKN1A蛋白表达水平均高于对照组(P<0.001);中、高剂量组TGF-β1 mRNA表达水平均高于对照组(P<0.001),TGF-β1蛋白表达水平随染毒剂量的增加而升高(P<0.001);CollagenⅢmRNA表达水平随染毒剂量的增加而升高(P<0.001),仅高剂量组CollagenⅢ蛋白表达水平[Background]The accumulation of inorganic arsenic in the liver can lead to liver fibrosis.Solute carrier family 7 member 11(SLC7A11)and cyclin dependent kinase inhibitor 1A(CDKN1A)are ferroptosis related genes that are abnormally expressed in liver and fibrosis diseases,but their impacts on inorganic arsenic induced liver fibrosis has not been reported yet.[Objective]To explore the effect of different doses of sodium arsenite on the migration ability of human hepatic stellate(LX-2)cells and the changes in DNA methylation and expression of SLC7A11 and CDKN1A in LX-2 cell activation induced by sodium arsenite.[Methods]LX-2 cells were treated with different doses of sodium arsenite for 24 h.The experiment set up four groups:control group(0μmol·L^(−1)),low-dose group(5μmol·L^(−1)),medium-dose group(10μmol·L^(−1)),and high-dose group(15μm·L^(−1)).Possible morphological changes of LX-2 cells were observed under an inverted microscope in each group.Cell migration and movement were determined by cell scratch assay.Mitochondrial morphology was observed in the control and the high-dose groups under a transmission electron microscope(TEM).Fe^(2+)fluorescence intensity was detected with a FerroOrange fluorescence probe.Differentially methylated sites were screened using the Illumina 850 K methylation chip.Ferroptosis related markers(SLC7A11&CDKN1A)and hepatic stellate cell activation related markers[transforming growth factor-β1(TGF-β1)and collagen typeⅢ(CollagenⅢ)]were detected by real-time fluorescence quantitative PCR and Western blot.[Results]With the increase of exposure dose,the cells retracted tentacles and gradually became round,and the healing of cell scratches gradually deteriorated.Destruction of mitochondrial membrane integrity and mitochondrial crista fracture in some cells were observed under a TEM in the high-dose group.Laser confocal microscopy showed that the fluorescence intensity of Fe^(2+)gradually increased with the increase of exposure dose.The results of methylation chip displayed

关 键 词：亚砷酸钠 人肝星状细胞 DNA甲基化 溶质载体家族7成员11 铁死亡 

分 类 号：R11[医药卫生—公共卫生与预防医学]