长链非编码核富含丰富的转录本1(lncRNA NEAT1)在妊娠合并系统性红斑狼疮患者中的表达及意义

Expression and significance of long noncoding RNA nuclear-enriched abundant transcript 1 (lncRNA NEAT1) in pregnant women with systemic lupus erythematosus

目的探讨妊娠合并系统性红斑狼疮(systemic lupus erythematosus,SLE)患者中长链非编码RNA核富含丰富的转录本1(long noncoding RNA nuclear-enriched abundant transcript 1,lncRNA NEAT1)介导的表观修饰调控辅助性T细胞2(Th2)分化发育的作用及其机制。方法选取2014年7月1日—2019年7月1日在河南省人民医院生产的正常单胎妊娠患者11例及妊娠合并SLE患者15例,收集外周血单个核细胞,qPCR检测NEAT1 mRNA表达水平。ELISA和流式细胞术检测IFN-γ和IL-4蛋白质表达水平。流式细胞术分选初始CD4^+T细胞,RNA结合蛋白免疫沉淀(RNA binding protein immunoprecipitation,RIP)技术检测组蛋白甲基转移酶(EZH2)与NEAT1结合情况。干扰NEAT1表达后,实时定量聚合酶链反应技术(real time quantity polymerase chain reaction,RT-qPCR)和蛋白质印迹法(Western blot)检测泛素E3连接酶(ITCH)的mRNA和蛋白质表达水平。运用染色质免疫沉淀(chromatin immunoprecipitation,ChIP)技术检测妊娠合并SLE患者EZH2在ITCH启动子区的募集。过表达NEAT1和ITCH,ELISA检测IL-4蛋白质水平。采用t检验进行数据统计学分析。结果妊娠合并SLE患者外周血NEAT1 mRNA水平显著高于正常妊娠对照组。妊娠合并SLE患者IFN-γ水平显著降低,IL-4水平显著上调。妊娠合并SLE患者初始CD4^+T细胞中,NEAT1与EZH2结合显著高于对照组。干扰NEAT1表达以后,ITCH的mRNA和蛋白质水平显著升高。ChIP分析发现,妊娠合并SLE患者中,EZH2在ITCH启动子区的募集也显著上调;ITCH能够显著抑制初始CD4^+T细胞中IL-4的产生,而过表达NEAT1则能够上调IL-4的蛋白质水平。结论妊娠合并SLE患者NEAT1水平显著升高,NEAT1招募EZH2到ITCH启动子区,促进初始CD4^+T细胞发育分化为Th2细胞,导致Th1/Th2失衡,从而影响妊娠合并SLE的疾病进程。

Objective To investigate the function and mechanism of long noncoding RNA nuclear-enriched abundant transcript 1(lncRNA NEAT1)-mediated epigenetic regulation of Th2 cell differentiation and development in pregnant women with systemic lupus erythematosus(SLE).Methods This study involved 11 women with normal singleton pregnancy(control group)and 15 pregnant women with SLE who delivered in the Henan Provincial People′s Hospital from July 1,2014 to July 1,2019.Peripheral blood mononuclear cells(PBMCs)were collected and analyzed by qPCR to detect the expression of NEAT1 at mRNA level.ELISA and flow cytometry were used to detect the expression of IFN-γand IL-4 at protein level.Naïve CD4^+T cells were sorted out by flow cytometry.RNA binding protein immunoprecipitation(RIP)was performed to detect the binding of EZH2 to NEAT1.After knockdown of NEAT1 expression,Real-time quantitative polymerase chain reaction(RT-qPCR)and Western blot were used to detect the expression of itchy E3 ubiguitin protein ligase(ITCH)at mRNA and protein levels.Chromatin immunoprecipitation(ChIP)was used to detect the abundance of EZH2 at ITCH promoter in pregnant patients with SLE.ELISA was used to detect IL-4 level after overexpression of NEAT1 and ITCH.Statistical data analysis was performed with t test.Results The expression of NEAT1 at mRNA level in peripheral blood of pregnant women with SLE was significantly higher than that in controls.IFN-γlevels were significantly reduced,while IL-4 levels were significantly increased in pregnant women with SLE than in controls.RIP analysis revealed that there was a great enrichment of NEAT1 in the naïve CD4^+T cells using anti-EZH2 compared to the control group.After knocking down the expression of NEAT1,the mRNA and protein levels of ITCH were significantly increased.ChIP assay demonstrated that EZH2 was recruited to the promoter of ITCH in pregnant women with SLE.ITCH significantly inhibited the production of IL-4 by naïve CD4^+T cells,while overexpression of NEAT1 upregulated the expression of IL-4 at protein level.Conclusions LncRNA NEAT1 was significantly up-regulated in pregnant women with SLE.It recruited EZH2 to the promoter of ITCH and promoted the differentiation of naïve CD4^+T cells to Th2 cells,resulting Th1/Th2 imbalance and affecting disease progression.