IGF_(2)BP_(1)调控lncRNA NEAT1在类风湿关节炎中的作用

Role of IGF_(2)BP_(1)in regulating lncRNA NEAT1 in rheumatoid arthritis

目的:类风湿关节炎(rheumatoid arthritis,RA)是一种常见的全身性自身免疫疾病,可能导致关节变形和功能障碍。本研究旨在探索胰岛素样生长因子-2 mRNA结合蛋白1(insulin like growth factor 2 mRNA binding protein 1,IGF_(2)BP_(1))在RA中的表达水平,以及其对长链非编码RNA(long non-coding RNA,lncRNA)核丰富转录本1(nuclear paraspeckle assembly transcript 1,NEAT1)稳定性的影响和在RA发病机制中的作用。方法:通过GEO(Gene Expression Omnibus)数据库获取RA数据集,并将其分为正常对照组和RA组,使用R软件分析获取N^(6)-甲基腺苷(N^(6)-methyladenosine,m^(6)A)相关的甲基化酶的表达量并进行差异分析。分析差异表达的m^(6)A甲基化酶与lncRNA NEAT1的相关性。通过在线网站ENCORI预测与lncRNA NEAT1结合的蛋白质,并与lncRNA NEAT1表达相关的m^(6)A甲基化酶取交集,从而获取关键基因。通过RNA蛋白质相互作用分析实验(RNA pull-down)验证在RA滑膜细胞中NEAT1与关键基因结合的情况。通过蛋白质印迹法验证关键基因在正常滑膜细胞和RA滑膜细胞中的表达水平。在RA滑膜细胞中转染关键基因的小干扰RNA,通过real-time RT-PCR检测NEAT1在滑膜细胞中的表达水平。结果:差异分析结果显示:与正常对照组相比,共有8个m^(6)A甲基化基因在RA组中存在差异表达,其中IGF_(2)BP_(1)、甲基转移酶样蛋白14(methyltransferase 14,N^(6)-adenosine-methyltransferase subunit,MEEEL14)与lncRNA NEAT1存在相关性;ENCORI预测结果显示:共有23个蛋白质能够与lncRNA NEAT1结合,与NEAT1共表达m^(6)A甲基化酶取交集,获得NEAT1相关m^(6)A甲基化蛋白IGF_(2)BP_(1)。蛋白质印迹法显示:与正常对照组相比,RA组中IGF_(2)BP_(1)蛋白在RA滑膜细胞中表达升高(P<0.05);RNA pull-down结果显示IGF_(2)BP_(1)蛋白与NEAT1结合;real-time RT-PCR的结果表明:敲减IGF_(2)BP_(1)可显著降低NEAT1 mRNA表达水平(P<0.01)。结论:IGF_(2)BP_(1)可能通过稳定lncRNA NEAT1表达参与RA发病,调控IGF_(2)BP_(1)水平可能是一种新的治疗RA的方法。

Objective:Rheumatoid arthritis(RA)is a prevalent systemic autoimmune disease that can lead to joint deformity and dysfunction.This study aims to explore the expression level of insulin-like growth factor 2 mRNA binding protein 1(IGF_(2)BP_(1))in RA,its impact on the stability of long noncoding RNA(lncRNA)nuclear paraspeckle assembly transcript 1(NEAT1),and its mechanism in RA pathogenesis.Methods:RA datasets were obtained from the Gene Expression Omnibus(GEO)database and categorized into a normal control group and a RA group.R software was used to analyze the expression of N^(6)-methyladenosine(m^(6)A)-related methyltransferases and differential analysis were performed.The correlation between differentially expressed m^(6)A methyltransferases and lncRNA NEAT1 was examined.Proteins predicted to bind with lncRNA NEAT1 were identified using the ENCORI online database,and key genes intersecting with m^(6)A methyltransferases associated with lncRNA NEAT1 expression were identified.RNA pull-down assays were used to verify the binding of NEAT1 with key genes in RA synovial cells.Western blotting was used to verify the expression levels of key genes in normal and RA synovial cells.After transfecting IGF_(2)BP_(1)small interfering RNA(siRNA)into RA synovial cells,the expression level of NEAT1 was detected by real-time RT-PCR.Results:Differential analysis showed that,compared to the normal control group,8 m^(6)Amethyltransferase genes were differentially expressed in the RA group,with IGF_(2)BP_(1)and methyltransferase 14,N^(6)-adenosine-methyltransferase subunit(MEEEL14)showing correlation with lncRNA NEAT1.ENCORI prediction results indicated that 23 proteins could bind to lncRNA NEAT1.The intersection with NEAT1 co-expressed m^(6)A methyltransferases identified IGF_(2)BP_(1)as the NEAT1-related m^(6)A methyltransferase.Western blotting revealed that IGF_(2)BP_(1)protein expression was significantly elevated in the RA group compared to the normal group(P<0.05).RNA pull-down assays demonstrated that IGF_(2)BP_(1)protein binds to NEAT1.Realtime RT-PCR results showed that knockdown of IGF_(2)BP_(1)significantly reduced NEAT1 mRNA expression levels(P<0.01).Conclusion:IGF_(2)BP_(1)may participate in RA pathogenesis by stabilizing lncRNA NEAT1 expression.Regulating IGF_(2)BP_(1)levels could be a novel therapeutic approach for RA.