雄黄主要成分二硫化二砷对骨髓增生异常综合征SKM-1细胞的甲基化作用效应  

Effect of Main Component of Realgar Arsenic Disulfide(As_(2)S_(2))on DNA Methylation of SKM-1 Cells with Myelodysplastic Syndrome

作　　者：丁小燕 王洪志[2] 张颖[1] 周庆兵[1] DING Xiao-yan;WANG Hong-zhi;ZHANG Ying;ZHOU Qing-bing(Institute of Geriatric Medicine,Xiyuan Hospital,China Academy of Chinese Medical Sciences,Beijing 100091,China;Institute of Hematology,Xiyuan Hospital,China Academy of Chinese Medical Sciences,Beijing 100091,China)

机构地区：[1]中国中医科学院西苑医院老年医学研究所,北京100091 [2]中国中医科学院西苑医院血液病研究所,北京100091

出　　处：《中国实验方剂学杂志》2022年第9期66-71,共6页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金青年基金项目(81603490);北京市自然科学基金青年项目(7174344);国家中医药管理局全国中医临床特色技术传承骨干人才培训项目(010070010)。

摘　　要：目的:从DNA甲基化角度探讨雄黄主要成分二硫化二砷(As_(2)S_(2))对骨髓增生异常综合征细胞株SKM-1细胞的效应机制。方法:细胞增殖与活性检测(CCK-8)试剂盒检测As2S2(0、1、2、4、8、16μmol·L^(-1))对SKM-1细胞的抑制作用;碘化丙啶(PI)染色法检测As2S2(0、1、2、4μmol·L^(-1))对SKM-1细胞周期的影响;运用人甲基化850K芯片在全基因组范围内观察As2S2(0、4μmol·L^(-1))对SKM-1细胞的甲基化效应并进行京都基因与基因组百科全书(KEGG)与基因本体(GO)分析;根据芯片数据,选取抑癌基因TUSC3,采用实时荧光定量聚合酶链式反应(Real-time PCR)与蛋白免疫印迹法(Western blot)技术观察As2S2(0、1、2、4μmol·L^(-1))对TUSC3 mRNA与蛋白的表达作用。结果:与空白组比较,As_(2)S_(2)对SKM-1细胞具有显著的抑制作用;增加G_(0)/G_(1)期细胞比例,降低S期细胞比例(P<0.05);850K芯片显示,4μmol·L^(-1)As_(2)S_(2)能显著引起SKM-1细胞基因组的甲基化变化,共有12710个差异甲基化基因(高甲基化与低甲基化基因各占50%),GO与KEGG数据库分析显示,差异甲基化基因涉及嘌呤代谢,自然杀伤细胞介导的增殖抑制作用,内吞作用,趋化因子信号通路,核泛素连接酶复合体等功能与通路;在下游基因表达方面,Real-time PCR与Western blot显示,与空白组比较,As_(2)S_(2)明显升高抑癌基因TUSC3的表达(P<0.05)。结论:雄黄主要成分As_(2)S_(2)对MDS细胞株SKM-1细胞具有显著的甲基化调控效应,并可能通过增加抑癌基因TUSC3的表达实现治疗效应。Objective:To explore the effects of the main component of Realgar arsenic disulfide(As_(2)S_(2))on DNA methylation of SKM-1 cells with myelodysplastic syndrome.Method:Cell Counting Kit-8(CCK-8)was used to detect the inhibitory effect of As_(2)S_(2)(0,1,2,4,8,16μmol·L^(-1))on SKM-1 cells.Propidium iodide(PI)staining was applied to detect the effect of As_(2)S_(2)(0,1,2,4μmol·L^(-1))on the SKM-1 cell cycle.The effect of As_(2)S_(2)(0,4μmol·L^(-1))on the methylation of SKM-1 cells on a genome-wide scale was observed by using Human Methylation 850K BeadChip,followed by Kyoto Encyclopedia of Genes and Genomes(KEGG)and gene ontology(GO)analyses.According to the microarray data,the antioncogene TUSC3 was selected,and real-time quantitative polymerase chain reaction(Real-time PCR)and Western blot were adopted to investigate the effect of As_(2)S_(2)(0,1,2,4μmol·L^(-1))on the mRNA and protein expression of TUSC3,respectively.Result:Compared with the conditions in the blank group,As_(2)S_(2)inhibited SKM-1 cells,increased the proportion of cells in the G_(0)/G_(1)phase,and decreased the proportion of cells in the S phase(P<0.05).The 850K microarray showed that 4μmol·L^(-1)As_(2)S_(2)could significantly induce DNA methylation in SKM-1 cells,with 12710 differentially methylated genes involved(50%hypermethylated and 50%hypomethylated genes).KEGG and GO analyses showed that differentially methylated genes were involved in many important biological functions and signaling pathways,including purine metabolism,natural killer cell-mediated cytotoxicity,endocytosis,chemokine signaling pathway,and nuclear ubiquitin ligase complex.In terms of downstream gene expression,Real-time PCR and Western blot showed that As_(2)S_(2)increased the expression of TUSC3,as compared with the conditions in the blank group(P<0.05).Conclusion:As_(2)S_(2),the main component of Realgar,has a significant regulatory effect on the methylation of SKM-1 cells,which is presumedly achieved by increasing the expression of TUSC3.

关 键 词：骨髓增生异常综合征 雄黄 DNA甲基化 抑癌基因 

分 类 号：R2-0[医药卫生—中医学] R22