Yang Ge, Wu Daoquan, Zeng Guang, Jiang Ou, Yuan Pingzong, Huang Shenjie, Zhu Jing, Tian Jie, Weng Yaguang, Rao Zhihua
Department of Clinical Laboratory, Affiliated Second People's Hospital of Luzhou Medical College Neijiang 641000, China.
Key Laboratory of Diagnostic Medicine Designated by Chinese Ministry of Education and School of Diagnostic Medicine, Chongqing Medical University Chongqing 400016, China.
Int J Clin Exp Pathol. 2015 Aug 1;8(8):8929-36. eCollection 2015.
It has been known that the occurrence of rheumatoid arthritis (RA) was closely correlated with DNA hypomethylation in CD4+ T cells, in which DNA methyltransferase plays a certain role. This study therefore investigated the effect of miR-126 on CD4+ T cell subgroup in RA patients and the alternation of DNA hypomethylation, in an attempt to provide new sights into the pathogenesis and treatment of RA. CD4+ T cells separated from RA patients were transfected with miRNA (miR)-126 expression vector or miR-126 inhibitor expression vector. The expression levels of CD11a, CD70 and DNMT1 mRNA were examined by real-time PCR. Protein levels of CD11a and CD70 were tested by flow cytometry while DNMT1 protein level was quantified by Western blotting. DNA was modified by sodium bisulfite and was sequenced for the methylation status of promoters of CD11a and CD70 genes. Both mRNA and protein expressions of CD11a and CD70 genes in CD4+ T cells were elevated by miR-126 transfection, along with decreased DNMT1 protein level but not mRNA level. The methylation degree of promoters of both CD11a and CD70 genes were significantly depressed after miR-126 transfection. The transfection by miR-126 inhibitor effectively reversed such effects. In RA patients, elevated miR-126 may promote the expression of CD11a and CD70 via the induction of hypomethylation of gene promoters by depressing DNMTI1 protein levels.
已知类风湿性关节炎（RA）的发生与CD4+T细胞中的DNA低甲基化密切相关，其中DNA甲基转移酶起一定作用。因此，本研究调查了miR-126对RA患者CD4+T细胞亚群的影响以及DNA低甲基化的变化，试图为RA的发病机制和治疗提供新的见解。将从RA患者中分离出的CD4+T细胞用miRNA（miR）-126表达载体或miR-126抑制剂表达载体进行转染。通过实时PCR检测CD11a、CD70和DNMT1 mRNA的表达水平。通过流式细胞术检测CD11a和CD70的蛋白水平，同时通过蛋白质印迹法定量DNMT1蛋白水平。DNA用亚硫酸氢钠修饰，并对CD11a和CD70基因启动子的甲基化状态进行测序。miR-126转染使CD4+T细胞中CD11a和CD70基因的mRNA和蛋白表达均升高，同时DNMT1蛋白水平降低但mRNA水平未降低。miR-126转染后，CD11a和CD70基因启动子的甲基化程度均显著降低。miR-126抑制剂转染有效逆转了这些作用。在RA患者中，升高的miR-126可能通过降低DNMTI1蛋白水平诱导基因启动子低甲基化，从而促进CD11a和CD70的表达。
