来源：风湿病与关节炎,2020,9（6）:6-11,20.

类风湿关节炎患者氧化应激关键lncRNAs表达谱的筛选验证及相关性分析（临床研究）

文建庭¹，刘　健²，万　磊²，姜　辉²，忻　凌²，孙　玥²，孙艳秋¹，张　颖¹，杜新雷¹，王　馨¹，王　杰¹

　　【摘　要】目的：检测类风湿关节炎患者外周血单个核细胞（PBMCs）氧化应激关键长链非编码RNA（lncRNAs）表达谱的差异，结合临床进行验证，筛选出类风湿关节炎氧化应激分子标志物，并进行相关性分析。方法：采用高通量测序技术分别对3例类风湿关节炎患者和3例健康人的PBMCs进行检测，基因聚类和KEGG通路分析类风湿关节炎氧化应激差异表达的lncRNAs，Cytoscape分析lncRNAs、miRNA、mRNA之间的共表达网络图；采用实时荧光定量PCR（qRT-PCR）对20例类风湿关节炎患者和20例健康人差异表达lncRNAs进行验证，ROC曲线分析lncRNAs作为类风湿关节炎氧化应激分子标志物的潜力，相关性分析lncRNAs与临床指标及患者感受（SPP）之间的关联。结果：①高通量测序结果显示，类风湿关节炎氧化应激相关的差异表达lncRNAs是34个，上调19个，下调15个，差异倍数最高的7条lncRNAs是AC019117.2、LINC00630、AC007952.5、LINC00663、LINC00638、MIAT、PSMG3-AS1；②生物信息学分析显示，氧化应激关键lncRNAs主要富集在weigel oxidative stress response，weigel oxidative stress

 by hne and H₂O₂等上；③RT-qPCR结果显示，与健康人相比，AC019117.2、LINC00630表达显著升高（P < 0.05），AC007952.5、LINC00663、LINC00638表达显著降低（P < 0.05），MIAT、PSMG3-AS1的表达差异无统计学意义（P > 0.05）；④ROC曲线结果显示，AC019117.2、LINC00630、AC007952.5、LINC00663、LINC00638曲线下面积（AUC）分别是0.676，0.784，0.834，0.643，0.638；⑥相关性分析结果显示，AC007952.5与超氧化物歧化酶（SOD）、年龄呈负相关，与免疫球蛋白呈正相关；AC019117.2与病程、疾病活动度（DAS28）、视觉模拟评分法（VAS）评分、焦虑量表（SAS）、抑郁量表（SDS）呈正相关，与生理功能（PF）、生理职能（RP）、躯体疼痛（BP）、精力（VT）、社会功能（SF）、情感职能（RE）呈负相关；LINC00638与类风湿因子、抗环瓜氨酸肽抗体、免疫球蛋白M、VAS评分呈正相关，与RP、一般健康状况、VT、SF呈负相关；LINC00663与病程呈正相关，与SOD、红细胞沉降率、C-反应蛋白呈负相关。结论：类风湿关节炎患者PBMCs存在氧化应激相关差异表达的lncRNAs，其中AC019117.2、LINC00630、AC007952.5、LINC00663、LINC00638有望成为类风湿关节炎氧化应激相关的分子标志物。

　　【关键词】　关节炎，类风湿；氧化应激；lncRNAs；患者感受

Screening and Correlation Analysis of lncRNAs Expression Profile of Oxidative Stress in Patients with Rheumatoid Arthritis

WEN Jian-ting,LIU Jian,WAN Lei,JIANG Hui,XIN Ling,SUN Yue,SUN Yan-qiu,ZHANG Ying,DU Xin-lei,WANG Xin,WANG Jie

　　【ABSTRACT】Objective:To detect the difference of lncRNAs expression profile of oxidative stress in PBMCs of patients with rheumatoid arthritis,and to screen out the molecular markers of oxidative stress and analyze the correlation.Methods:The PBMCs of three patients with rheumatoid arthritis and three healthy people were detected by high-throughput sequencing.Gene clustering and KEGG Pathway were used to analyze lncRNAs;Cytoscape was used to analyze the network diagram co-expressed between lncRNAs,miRNA and mRNA.qRT-PCR was used to verify the differential expression of lncRNAs in 20 patients with rheumatoid arthritis and 20 healthy people.ROC curve was used to analyze the potential of lncRNAs as a molecular marker of oxidative stress in rheumatoid arthritis.Correlation between lncRNAs and clinical indexes and SPP was analyzed.Results:①The results of high-throughput sequencing showed that there were 34 differential expressions of lncRNAs,among whcih 19 were up-regulated and 15 were down regulated,and the 7 lncRNAs with the highest multiple of difference were AC019117.2,LINC00630,AC007952.5,LINC00663,LINC00638,MIAT and PSMG3-AS1;②Bioinformatics analysis showed that lncRNAs of oxidative stress were mainly enriched in weigel oxidative stress response,weigel oxidative stress by hne and H₂O₂;③The results of RT-qPCR showed that compared with healthy people,the expressions of AC019117.2 and LINC00630 significantly increased（P < 0.05）,the expressions of AC007952.5,LINC00663 and LINC00638 significantly decreased（P < 0.05）,and the expression differences of MIAT and PSMG3-AS1 had no statistical significance（P > 0.05）;④The results of ROC curve showed that the AUC of AC019117.2,LINC00630,AC007952.5,LINC00663,LINC00638 were 0.676,0.784,0.834,0.643 and 0.638 respectively;⑤The results of correlation analysis showed that AC007952.5 was negatively correlated with SOD and age,and positively correlated with immunoglobulin;AC019117.2 was positively correlated with DAS28,VAS,SAS and SDS and negatively correlated with PF,RP,BP,VT,SF and RE;LINC00638 was positively correlated with rheumatoid factor,anti cyclic citrullinated peptide antibody,immunoglobulin M and VAS,and negatively correlated with RP,general health status,VT,SF;LINC00663 was positively correlated with the course of disease,and negatively correlated with SOD,erythrocyte sedimentation rate,C-reactive protein.Conclusion:There are lncRNAs related to oxidative stress in PBMCs of patients with rheumatoid arthritis,among which AC019117.2,LINC00630,AC007952.5,LINC00663,LINC00638 are expected to be molecular markers related to oxidative stress in rheumatoid arthritis.

　　【Keywords】 arthritis,rheumatoid;oxidative stress;lncRNAs;SPP