类风湿关节炎成纤维样滑膜细胞lncRNA-miRNA-mRNA网络构建及关键基因预测

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摘要目的探索类风湿关节炎（RA）成纤维样滑膜细胞（FLS）中长链非编码RNA（lncRNA）-微小RNA（miRNA）-mRNA网络及关键lncRNA，揭示lncRNA在RA中的作用。方法在美国国立生物技术信息中心的基因表达数据库（GEO）中获取相关的数据集GSE103578、GSE83147、GSE128813，每个数据集中对照组含有3个健康人滑膜分离的FLS，RA组含有3个RA患者的FLS。使用R语言程序，idmap3包进行探针注释，limma包对数据进行标准化、去除批次效应及基因差异表达分析，以logFC＞1.0且P ＜ 0.05作为差异表达的标准。对差异表达的lncRNA及mRNA进行相关性分析，在lncbase、starBase、miRcode数据库中，获得与lncRNA相关的miRNA，在miRWalk、TargetScan数据库中寻找与mRNA相关的miRNA，二者取交集，通过Cytoscape软件，构建lncRNA-miRNA-mRNA网络，将网络中的lncRNA作为关键lncRNA。结果 GEO数据集进行标准化、去除批次效应及注释后，最终得到mRNA有14 760条，lncRNA有5290条。与对照组比较，RA组上调的mRNA有31条，下调56条，lncRNA中上调的基因有9条，下调14条。以Pearson相关系数R > 0.85且P ＜ 0.05作为lncRNA-mRNA共表达基因的标准，得到共表达lncRNA有18条，mRNA有51条。与共表达的lncRNA、mRNA有结合位点的miRNA有26条。构建的lncRNA-miRNA-mRNA共表达网络中lncRNA有13条，包括lncRNA XIST、NR2F2-AS1、LINC01018等，miRNA有26条，mRNA有46条。结论 lncRNA XIST、NR2F2-AS1、LINC01018等13条lncRNA在RA-FLS中差异表达，并通过竞争内源性RNA机制调节miRNA及mRNA的表达，形成lncRNA-miRNA-mRNA调节网络，可作为进一步验证的目标lncRNA。

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关键词 ：类风湿关节炎, 成纤维样滑膜细胞, 长链非编码RNA, 竞争内源性RNA

Abstract：Objective To explore the long non-coding RNA (lncRNA) -microRNA (miRNA) -mRNA network and key lncRNA in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS), and to reveal the role of lncRNA in RA. Methods Data sets GSE103578, GSE83147 and GSE128813 were obtained from Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information in the United States. Each data set contained three synovium-isolated FLS from healthy people in the control group and three FLS from RA patients in the RA group. R language program, idmap3 package was used for probe annotation. Limma package was used to standardize the data, remove batch effect and analyze gene differential expression. LogFC > 1.0 and P < 0.05 were used as the criteria for differential expression. Correlation analysis was conducted for the differentially expressed lncRNA and mRNA. In the lncbase, starBase and miRcode databases, the miRNAs related to lncRNA were obtained, and the miRNAs related to mRNA were searched in the miRWalk and TargetScan databases. The intersection of the two was taken, and the lncRNA-miRNA-mRNA network was constructed by Cytoscape software, and the lncRNAs in the network were taken as the key lncRNA. Results After standardization of GEO data set, elimination of batch effect and annotation, 14 760 mRNAs and 5290 lncRNAs were finally obtained. Compared with the control group, there were 31 mRNA up-regulated and 56 mRNA down-regulated in RA group, and 9 up-regulated genes and 14 down-regulated genes in lncRNA. The Pearson correlation coefficient R > 0.85 and P < 0.05 were used as the criteria for the co-expression of lncRNA-mRNA genes, and 18 co-expressed lncRNAs and 51 mRNAs were obtained. There were 26 miRNAs that had binding sites with co-expressed lncRNA and mRNA. In the constructed lncRNA-miRNA-mRNA co-expression network, there were 13 lncRNAs, including lncRNA XIST, NR2F2-AS1, LINC01018, etc., 26 miRNAs and 46 mRNAs. Conclusion Thirteen lncRNAs, such as lncRNA XIST, NR2F2-AS1, LINC01018, are differentially expressed in RA-FLS, and the expression of miRNA and mRNA is regulated through the competitive endogenous RNA mechanism to form a regulatory network of lncRNA-miRNA-mRNA, which could be used as the target lncRNA for further verification.

Key words： Rheumatoid arthritis Fibroblast-like synoviocytes long non-coding RNA Competitive endogenous RNA

基金资助:国家自然科学基金资助项目（81774114）。

通讯作者: 何东仪（1967.9-），男，博士，教授，主任医师，博士生导师；研究方向：中西医结合治疗类风湿关节炎。

作者简介: 周新朋（1989.3-），男，上海中医药大学附属光华医院2016级中西医结合临床专业在读博士研究生；研究方向：中西医结合治疗类风湿关节炎。

引用本文:

周新朋1,2 金晔华1,2 张润润1,2 何东仪1,2. 类风湿关节炎成纤维样滑膜细胞lncRNA-miRNA-mRNA网络构建及关键基因预测[J]. 中国医药导报, 2021, 18(9): 12-16.
ZHOU Xinpeng1,2 JIN Yehua1,2 ZHANG Runrun1,2 HE Dongyi1,2. Construction of lncRNA-miRNA-mRNA network and prediction of key genes in rheumatoid arthritis fibroblast-like synoviocytes. 中国医药导报, 2021, 18(9): 12-16.

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