强直性脊柱炎患者H3K27me3表达水平及其调控Th17细胞分化的表观遗传机制

H3K27me3 Expression Level and Its Epigenetic Regulation Mechanisms in Th17 Cell Differentiation in Patients With Ankylosing Spondylitis

目的 探讨强直性脊柱炎(ankylosing spondylitis, AS)中组蛋白H3K27me3甲基化及其调节酶JMJD3和EZH2在Th17细胞分化中的作用,以揭示其在AS发病机制中的潜在作用。通过分析H3K27me3的甲基化状态及其与Th17相关因子之间的相互作用,为AS的临床治疗提供新的策略和靶点。方法 采集84名AS的患者(活跃期、稳定期各42例)和84名健康志愿者(对照组)血液样本,ELISA检测Th17细胞及相关细胞因子(IL-21、IL-22和IL-17),RT-PCR分析Th分化关键信号通路RORc、JAK2、STAT3基因的表达,蛋白质印迹法检测RORc、JAK2/STAT3通路蛋白、H3K27me3及相关蛋白酶EZH2、JMJD3的表达。对活动期AS患者H3K27me3、EZH2、JMJD3与Th分化关键信号通路分子的关联性进行Pearson相关分析。结果 RORc、JAK2、STAT3 mRNA表达,活动期组>稳定期组,差异有统计学意义(P<0.05)。H3K27me3、EZH2的表达量,活动期组<稳定期组<对照组,差异有统计学意义(P<0.05);JMJD3、RORc、JAK2、pJAK2、STAT3、pSTAT3的表达量,活动期组>稳定期组>对照组,差异有统计学意义(P<0.05)。活动期组Th17比例及其炎性因子的表达水平高于其他两组(P<0.05)。H3K27me3与RORc、JAK2、STAT3、IL-17负相关,JMJD3与JAK2、STAT3、IL-17正相关,而EZH2与JAK2、STAT3、IL-17负相关(P<0.05)。结论 AS中H3K27me3的低表达受到基因位点JMJD3和EZH2的影响,可以调节Th17细胞的分化,从而在AS的发病和进展中发挥作用。

Objective To investigate the roles of histone H3K27me3 methylation and its regulatory enzymes JMJD3 and EZH_(2) in the differentiation of Th17 cells in ankylosing spondylitis(AS),to unveil their potential involvement in the pathogenesis of AS,and to provide new strategies and targets for the clinical treatment of AS by analyzing the methylation state of H3K27me3 and its interactions with Th17-related factors.Methods A total of 84 AS patients(42 active AS patiens and 42 patients in the stable phase of AS)were enrolled for the study,while 84 healthy volunteers were enrolled as the controls.Blood samples were collected.Peripheral blood mononuclear cells were isolated.ELISA assay was performed to examine Th17 cells and the relevant cytokines IL-21,IL-22,and IL-17.The mRNA expressions of RORc,JAK2,and STAT3 were analyzed by RT-PCR,the protein expressions of RORc,JAK2/STAT3 pathway protein,H3K27me3 and the relevant protease(EZH_(2) and JMJD3)were determined by Western blot.Correlation between H3K27me3,EZH_(2) and JMJD3 and the key signaling pathway molecules of Th cell differentiation was analyzed by Pearson correlation analysis.Results The mRNA expressions of RORc,JAK2,and STAT3 were significantly higher in the active phase group than those in the stable phase group(P<0.05).The relative grayscale values of H3K27me3 and EZH_(2) in the active phase group were lower than those of the stable phase group,which were lower than those of the control group,with the differences being statistically significant(P<0.05).The relative grayscale values of JMJD3,RORc,JAK2,pJAK2,STAT3,and pSTAT3 proteins were significantly higher in the active phase group than those in the stable phase group,which were higher than those in the control group(all P<0.05).The proportion of Th17 and the expression level of inflammatory factors in the active period group were higher than those in the other two groups(P<0.05).H3K27me3 was negatively correlated with RORc,JAK2,STAT3,and IL-17,JMJD3 was positvely correlated with JAK2,STAT3,and IL-17,and EZH_(2) was negatively correlated with JAK2,STAT3,and IL-17(all P<0.05).Conclusion The low expression of H3K27me3 in AS is influenced by the gene loci JMJD3 and EZH_(2),which can regulate the differentiation of Th17 cells and thus play a role in the pathogenesis and progression of AS.