类风湿关节炎滑膜成纤维样细胞自噬相关长链非编码RNA筛选和分析  

Screening and analysis for autophagy-related lncRNA in fibroblast-like synoviocytes from patients with rheumatoid arthritis

作　　者：尚华 彭清[1] 刘佳君 刘焱 龙丽 SHANG Hua;PENG Qing;LIU Jiajun;LIU Yan;LONG Li(Zunyi Medical University,Zunyi Guizhou 563000;Department of Rheumatologyand Immunology,Suining Central Hospital,Suining Sichuan 629000;Department of Rheumatology and Immunology,Sichuan Provincial People's Hospital,Chengdu 610072,China)

机构地区：[1]遵义医科大学,贵州遵义563000 [2]遂宁市中心医院风湿免疫科,四川遂宁629000 [3]四川省人民医院风湿免疫科,成都610072

出　　处：《中南大学学报（医学版）》2021年第10期1071-1079,共9页Journal of Central South University ：Medical Science

摘　　要：目的:长链非编码RNA(long non-coding RNA,lncRNA)已成为调控基因表达和影响多种生物学过程的关键表观遗传调控因子。在类风湿关节炎(rheumatoid arthritis,RA)的发生发展中,lncRNA发挥着重要作用,有关lncRNA在RA患者外周血细胞中的研究已有报道。然而,目前尚无lncRNA在RA患者中对于自噬调控的研究。本研究通过筛选RA滑膜成纤维样细胞(rheumatoid arthritis fibroblast-like synoviocytes,RA-FLSs)自噬前后差异表达的lncRNAs,寻找调节RA-FLSs自噬的关键lncRNAs,旨在为RA的诊断和治疗提供新方向。方法:获取6例RA患者膝关节或髋关节术后的滑膜组织,原代培养(组织块法)RA-FLSs至第5代,用mTOR抑制剂PP242处理后,通过蛋白质印迹法检测细胞自噬水平标记蛋白LC3-II表达水平的变化。其中3例采用TRIzol提取PP242处理前后RA-FLSs总RNA,应用Agilent Human ceRNA Microarray 2019(4×180 K,Design ID:086188)芯片检测,发现表达量显著改变的lncRNAs(差异倍数≥2.0,P<0.05)。利用生物信息学技术,对获得的差异表达的lncRNAs进行基因本体(gene ontology,GO)分析和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路分析,以发现差异表达的lncRNAs可能参与的生物学通路,探索差异表达的lncRNAs在RA发病机制中的作用。在前期芯片筛选出的lncRNAs差异表达的基础上,结合GO富集分析和KEGG分析,筛选出ENST00000584721.1、ENST00000615939.1,对全部6例RA-FLSs进行real-time RT-PCR验证。结果:从患者膝关节或髋关节滑膜组织中成功分离、培养出RAFLSs。6例RA-FLSs经过PP242处理后,自噬水平标记蛋白LC3-II表达水平升高(P<0.05),PP242诱导RA-FLSs发生自噬。芯片筛选共有591个差异表达的lncRNAs,其中表达上调的有428个,表达下调的有163个。GO分析显示这些差异表达的lncRNAs与Th17功能、T细胞相关细胞因子生成及TGF-β受体信号通路的负性调节,以及I型TGF-β受体结合和IL-6受体复�Objective: Long non-coding RNA(lncRNA) has become a key epigenetic regulator that regulates gene expression and affects a variety of biological processes. LncRNA plays an important role in the occurrence and development of rheumatoid arthritis(RA). The study on lncRNA in peripheral blood cells of RA patients has been reported. However, there is no study on autophagy regulation by lncRNA in RA patients. This study aims to provide a new direction for the diagnosis and treatment of RA via screening the changes of lncRNAs in RA fibroblast-like synoviocytes(RA-FLSs) before and after autophagy and finding the key lncRNAs targeting RA-FLSs autophagy.Methods: Synovial tissues of 6 RA patients after knee and hip joint surgery were obtained,and RA-FLSs were cultured to the 5 th generation for further experiments(tissue culture method). After treatment with mTOR inhibitor PP242, the expression of LC3-II was detected by Western blotting. Total RNAs of 3 cases of RA-FLSs before and after treatment with mTOR inhibitor PP242 were extracted by TRIzol and screened by Agilent Human ce RNA Microarray 2019(4×180 K, design ID: 086188) chip. The lncRNAs with significantly changed expression levels were selected(difference multiple≥2.0, P<0.05).Bioinformatics technology was used to analyze the gene ontology(GO) function and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway of the differentially expressed lncRNAs, and to explore the possible role of differentially expressed lncRNAs in the pathogenesis of RA. Subsequently, ENST00000584721.1 and ENST00000615939.1 were identified in all the 6 samples of RA-FLSs using real-time RT-PCR, which were selected by previous chip screen combined with GO enrichment analysis and KEGG analysis.Results: RA-FLSs were successfully isolated and cultured from the synovial tissues of the patient’s knee or hip joint. After 6 RA-FLSs were treated with PP242, the expression level of autophagy marker protein LC3-II was increased(P<0.05). PP242 induced autophagy in RA-FLSs. LncRNA sequencing analysis sh

关 键 词：类风湿关节炎 滑膜成纤维样细胞 长链非编码RNA 自噬 芯片 

分 类 号：R593.22[医药卫生—内科学]