HMGB1/GADD45A介导急性移植物抗宿主病患者CD4^+T细胞STAT3启动子的去甲基化  

Regulation of DNA demethylation of STAT3 promoter in CD4^+ T cells from aGVHD patients by HMGB1/GADD45A

作　　者：徐雅靖[1] 杨晶 张媛媛[1] 刘恩伊[1] 彭捷[1] 陈旭[1] 陈方平[1] 彭敏源[1] XU Yajing, YANG Jing, ZHANG Yuanyuan, LIU Enyi, PENG Jie, CHEN Xu, CHEN Fangping, PENG Minyuan(Department of Hematology, Xiangya Hospital, Central South University, Changsha 410008, Chin)

机构地区：[1]中南大学湘雅医院血液科,长沙410008

出　　处：《中南大学学报（医学版）》2018年第9期937-944,共8页Journal of Central South University ：Medical Science

基　　金：国家自然科学基金(81570165)~~

摘　　要：目的:研究急性移植物抗宿主病(acute graft-versus-host disease,a GVHD)患者CD4+T细胞中STAT3基因启动子DNA病理性低甲基化的分子调控机制。方法:收集行同胞全相合异基因造血干细胞移植的42例患者的血液样本。采用实时定量PCR和Western印迹检测各组患者生长阻滞与DNA损伤诱导蛋白45A(growth arrest and DNA damage-inducible protein 45 alpha,GADD45A)的表达水平,免疫共沉淀检测各组患者高迁移率族蛋白1(high mobility group box-1 protein,HMGB1)与GADD45A的结合水平,染色质免疫共沉淀检测各组患者HMGB1和GADD45A与STAT3启动子的结合水平;正常CD4+T细胞中过表达HMGB1及干扰GADD45A表达后用Western印迹检测STAT3表达水平,亚硫酸盐测序检测STAT3启动子区DNA甲基化水平。结果:与未发生a GVHD患者比较,a GVHD患者外周血CD4+T细胞中GADD45A表达水平明显升高;HMGB1与GADD45A的结合显著增加;HMGB1,GADD45A与STAT3启动子的结合水平均明显升高,且STAT3启动子区HMGB1,GADD45A结合水平与其DNA甲基化水平呈高度负相关(分别r=0.719,r=0.840;P<0.01)。与单纯过表达HMGB1相比,正常CD4+T细胞中过表达HMGB1叠加干扰GADD45A表达后STAT3表达明显降低,STAT3启动子DNA甲基化水平明显升高。结论:CD4+T细胞中HMGB1和GADD45A表达升高是异基因造血干细胞移植后患者STAT3启动子DNA低甲基化,STAT3高表达,进而诱发a GVHD的重要因素。Objective： To study the molecular mechanism for DNA hypomethylation of STAT3 promoter in CD4^＋ T cells from acute graft -versus-host disease （aGVHD） patients.Methods： We collected CD4^＋ T cells from peripheral blood of 42 patients who underwent allogeneic hematopoietic stem cell transplantation （allo-HSCT） from HLA-identical sibling donors. GADD45A expression level in CD4^＋ T cells was measured by real-time PCR and Western blot. The binding level between HMGB1 and GADD45A in CD4^＋ T cells was analyzed by coimmunoprecipitation, while the binding levels of HMGB1/GADD45A with STAT3 promoter were detected by chromatin immunoprecipitation-quantitative real-time PCR （ChIP-qPCR）. After overexpression of HMGB1 and knockdown of GADD45A in normal CD4^＋ T cells, STAT3 expression and DNA methylation were measured by Western blot and bisulfite sequencing PCR,respectively.Results： GADD45A expression was significantly up-regulated in patients with aGVHD compared with that in the patients without aGVHD. More HMGB1-GADD45A complexes were found in CD4^＋ T cells from patients with aGVHD compared with that in patients without aGVHD. The bindings of HMGB1/GADD45A with STAT3 promoter were significantly increased, and the binding levels of HMGB1/GADD45A were negatively correlated with STAT3 promoter DNA methylation. The expression of STAT3 was significantly reduced and the DNA methylation of STAT3 promoter was significantly increased in CD4^＋ T cells with overexpression of HMGB1 and knockdown of GADD45A compared with CD4^＋ T cells only with overexpression of HMGB1. Conclusion： The increased expression of HMGB1/GADD45A plays an importent role in STAT3 promoter DNA hypomethylation, thereby promoting STAT3 expression in CD4^＋ T cells from aGVHD patients.

关 键 词：急性移植物抗宿主病 DNA甲基化 高迁移率族蛋白1 生长阻滞与DNA损伤诱导蛋白45A STAT3 

分 类 号：R457.7[医药卫生—治疗学]