摘要
目的探究亚砷酸钠诱导的活性氧是否影响N~6-甲基腺苷(m~6A)水平,从RNA表观遗传学角度探讨亚砷酸钠的毒性机制。方法以亚砷酸钠作为活性氧诱导剂,以N-乙酰半胱氨酸作为活性氧清除剂,检测细胞在亚砷酸钠单独处理和亚砷酸钠联合N-乙酰半胱氨酸处理情况下皮肤细胞活性氧积累情况,同时测定m~6A及其甲基化酶的m RNA和蛋白水平的改变。结果在5、10和15μM的亚砷酸钠处理下,活性氧含量随砷浓度升高而增加;在亚砷酸钠染毒浓度为15μM时,细胞内总m~6A水平水平相比于对照组升高了1.49倍(P<0.05),m~6A甲基化酶(METTL3、METTL14和WTAP)m RNA表达水平相应升高(P<0.05),METTL14和WTAP蛋白表达水平分别为对照组的1.47倍和1.85倍(P<0.05)。N-乙酰半胱氨酸可以降低异常升高的活性氧、m~6A以及其甲基化酶水平。结论亚砷酸钠诱导的活性氧能增加人皮肤细胞m~6A的修饰水平,其机制可能与上调m~6A甲基化酶表达水平有关。
Objective The aim of this study was to explore whether reactive oxygen species(ROS) induced by sodium arsenite affect N~6-methyladenosine(m~6A) level, in order to provide a new idea for the study on toxicity induced by arsenite from the perspective of RNA epigenetics. Methods Arsenite was used to induce ROS, and N-acetylcysteine was used to eliminate ROS. ROS level, mRNA and protein levels of m~6A and its methylases(METTL3, METTL14, and WTAP) were tested in arsenite treatment group and arsenite combined with N-acetylcysteine treatment group. Results 5, 10 and 15 μM of arsenite elevated ROS level in a concentration-dependent manner. With the treatment of 15 μM arsenite, the m~6A level increased 1.49-fold compared to the control(P<0.05), the RNA levels of m~6A methylases(METTL3, METTL14 and WTAP) elevated(P<0.05), and the protein levels of METTL14 and WTAP were increased 1.47-fold and 1.85-fold compared to the control,respectively(P<0.05). N-acetylcysteine could suppress abnormal increase of m~6A and its methylases levels in HaCaT cells treated by arsenite. Conclusion ROS induced by arsenite increases m~6A level possibly via upregulation of m~6A methylases.
引文
[1]Valko M,Jomova K,Rhodes CJ,et al.Redox-and non-redoxmetal-induced formation of free radicals and their role in human disease[J].Archives of Toxicology,2015,90(1):1-37.
[2]Afanas'ev I.New nucleophilic mechanisms of ros-dependent epigenetic modifications:comparison of aging and cancer[J].Aging and Disease,2013,5(1):52-62.
[3]Maity A,Das B.N6-methyladenosine modification in m RNA:machinery,function and implications for health and diseases[J].FEBSJournal,2016,283(9):1607-1630.
[4]代黄梅,谷仕艳,李欣洋,等.N6-甲基腺苷在三氧化二砷抑制肝癌细胞生长中的作用研究[J].现代预防医学,2018,45(8):1471-1474,1486.
[5]Shen L,Song CX,He C.Mechanism and function of oxidative reversal of DNA and RNA methylation[J].Annual Review of Biochemistry,2014,83:585-614.
[6]Dominissini D,Moshitch-Moshkovitz S,Schwartz S,et al.Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq[J].Nature,2012,485(7397):201-206.
[7]Fu Y,Dominissini D,Rechavi G,et al.Gene expression regulation mediated through reversible m(6)A RNA methylation[J].Nature Reviews Genetics,2014,15(5):293-306.
[8]Ping XL,Sun BF,Wang L,et al.Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase[J].Cell Research,2014,24(2):177-189.
[9]Zou S,Toh JD,Wong KH,et al.N(6)-Methyladenosine:a conformational marker that regulates the substrate specificity of human demethylases FTO and ALKBH5[J].Scientific Reports,2016,6:25677.
[10]Costa M.The control of histone methylation and gene expression by oxidative stress,hypoxia and metals[J].Free Radical Biology and Medicine,2011,51(Supplement 1):S3.
[11]Wu QH,Ni XH.ROS-Mediated DNA methylation pattern alterations in carcinogenesis[J].Current Drug Targets,2015,16(1):13-19.
