类风湿性关节炎成纤维样滑膜细胞特征基因TNFAIP6的鉴定及验证  

Identification and validation of hub gene TNFAIP6 of fibroblast-like synoviocytes in rheumatoid arthritis

作　　者：雷蕾 吕玉欣 张晶[1] 陈陵 LEI Lei;LYU Yuxin;ZHANG Jing;CHEN Ling(Department of Hematology,Army Medical Center of PLA/Daping Hospital of Third Military Medical University,Chongqing;Department of Diabetic Ophthalmology,Eye Hospital of China Academy of Chinese Medical Sciences,Beijing;Department of Gastroenterology,No.958 Hospital of PLA Army,Chongqing;Department of Gastroenterology,Xiji Hospital,Chongqing,China)

机构地区：[1]陆军特色医学中心(第三军医大学大坪医院)血液内科,重庆 [2]中国中医科学院眼科医院糖尿病眼病科,北京 [3]陆军第958医院消化内科,重庆 [4]重庆西计医院消化内科,重庆

出　　处：《陆军军医大学学报》2025年第3期234-242,共9页Journal of Army Medical University

基　　金：重庆市自然科学基金面上项目(cstc2020jcyj-msxmX1055)。

摘　　要：目的鉴定类风湿性关节炎(rheumatoid arthritis,RA)成纤维样滑膜细胞的特征基因,并验证其对增殖、迁移、侵袭能力的影响。方法从基因表达综合(Gene Expression Omnibus,GEO)数据库下载4个独立的滑膜组织微阵列转录组测序数据集(GSE1919、GSE55235、GSE55457、GSE77298)和1个滑膜细胞单细胞测序数据集(GSE192504),利用差异基因分析、加权基因共表达网络分析(weighted gene co-expression network analysis,WGCNA)、单细胞测序数据分析等多种生物信息学分析方法鉴定RA成纤维样滑膜细胞的特征基因。利用RT-qPCR和Western blot检测肿瘤坏死因子α诱导蛋白6(TNF alpha induced protein 6,TNFAIP6)在人RA成纤维样滑膜细胞系MH7A体外模拟炎症环境下的表达水平。MH7A转染si-TNFAIP6进行基因沉默,采用CCK-8和Transwell实验检测沉默TNFAIP6对MH7A细胞增殖、迁移、侵袭能力的影响。结果联合差异基因分析、WGCNA和单细胞测序数据分析,确定了1个RA成纤维样滑膜细胞特征基因(TNFAIP6)。RT-qPCR和Western blot显示,与无菌磷酸盐缓冲液(phosphate buffered saline,PBS)处理比较,10 ng/mL浓度的肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)显著上调MH7A细胞TNFAIP6的mRNA和蛋白表达(P<0.05)。CCK-8和Transwell实验显示,在MH7A细胞敲低TNFAIP6的表达后,敲低组(si-TNFAIP6)的细胞增殖、迁移和侵袭能力相较于对照组(FAM-si-NC)有显著的降低(P<0.05)。结论TNFAIP6在RA成纤维样滑膜细胞中特异性高表达,促进其增殖、迁移、侵袭能力,可能是RA成纤维样滑膜细胞异常活化的主要贡献者。Objective To identify the hub gene of fibroblast-like synoviocytes in rheumatoid arthritis(RA)and verify its impact on proliferation,migration and invasion of the cells.Methods Four independent synovial tissue microarray transcriptome sequencing datasets and one single cell sequencing dataset were downloaded from the Gene Expression Omnibus(GEO)database,and the hub gene of RA fibroblast-like synovial cells was identified by differential gene analysis,weighted gene co-expression network analysis(WGCNA)and single-cell sequencing data analysis.The expression of TNFAIP6 in human RA fibroblast-like synovial cell line MH7A was detected by RT-qPCR and Western blotting under simulated inflammatory environment.After MH7A cells were transfected with si-TNFAIP6,CCK-8 and Transwell assays were applied to detect the effects of silencing TNFAIP6 on the proliferation,migration and invasion abilities of MH7A cells.Results With the aid of differential gene expression analysis,WGCNA,and single-cell sequencing data,we identified TNFAIP6 as the characteristic gene of RA fibroblast-like synoviocytes.RT-qPCR and Western blot assay demonstrated that TNFAIP6 was significantly highly expressed at mRNA and protein levels in MH7A cells stimulated with 10 ng/mL TNF-αwhen compared to the cells treated with PBS(P<0.05).CCK-8 and Transwell assays indicated that the silencing of TNFAIP6 markedly inhibited the proliferation,migration and invasion abilities of MH7A cells when compared with the control cells(FAM-si-NC,P<0.05).Conclusion TNFAIP6 is highly expressed specifically in RA fibroblast synoviocytes,which promotes the proliferation,migration and invasion,and may be the main contributor to the abnormal activation of RA fibroblast synoviocytes.

关 键 词：类风湿性关节炎 成纤维样滑膜细胞 肿瘤坏死因子α诱导蛋白6 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学] R394.3[医药卫生—基础医学] R593.22