基于转录组学分析CD146在人类风湿关节炎成纤维样滑膜细胞条件培养液状态下的EAhy.926细胞中的基因表达差异

Analyzing Differential Gene Expression of CD146 in EAhy.926 Cultured in Human Rheumatoid Arthritis Fibroblast Like Synovial Cell Conditioned Medium Based on Transcriptome Sequencing Technology

黑色素瘤细胞黏附分子CD146作为一种新的内皮细胞标志物,其表达与肿瘤、自身免疫性疾病的进展密切相关。类风湿关节炎(rheumatoid arthritis,RA)是一种以对称性、慢性多关节炎症为特征表现的系统性自身免疫性疾病,滑膜血管翳作为RA的代表性病理产物在疾病的发生发展中起着重要作用。为了研究CD146分子在人类风湿关节炎成纤维样滑膜细胞(human rheumatoid arthritis fibroblast like synovial cell,HFLS-RA)条件培养液状态下的人脐静脉内皮细胞株EAhy.926的基因表达差异及其影响机制,本研究通过慢病毒介导的RNA干扰技术抑制EAhy.926细胞中CD146的表达。培养并提取HFLS-RA上清液制备条件培养液。将转染后的EAhy.926细胞用HFLS-RA条件培养液培养24 h构建RA损伤模型,并对其进行转录组测序,筛选出差异表达基因(differentiallly expressed genes,DEGs),并通过GO、KEGG通路分析,探索CD146调控的DEGs主要参与的相关信号通路及生物学过程。结果显示,与对照组细胞相比,HFLS-RA条件培养液状态下CD146敲低的EAhy.926细胞有341个DEGs,其中上调基因有173个,下调基因有168个;GO、KEGG通路分析发现DEGs主要富集在Wnt信号通路、PPAR信号通路、TRP通道的炎症介质调节、脂肪细胞因子信号通路上,参与了细胞黏附、细胞运动、细胞定位、半胱氨酸生物合成过程等生物过程。本研究通过转录组学分析发现在HFLS-RA条件培养液状态下,CD146分子可能通过多条关键信号通路及多个基因影响EAhy.926。研究结果为探讨CD146分子在类风湿关节炎滑膜血管生成中的作用及机制的研究提供了理论依据。

As a new endothelial cell marker,the expression of melanoma cell adhesion molecule,CD146,has been found to be closely related to the progression of tumors and autoimmune diseases.Rheumatoid arthritis(RA)is a systemic autoimmune disease characteri-zed by symmetrical and chronic polyarthritis.Synovial pannus,as a representative pathological product of RA,plays an important role in the occurrence and development of the disease.In order to investigate the differential gene expression and influencing mechanism of CD146 in human unbilical vein endothelial cell line,EAhy.926,under the condition of human rheumatoid arthritis fibroblast like synovial cell(HFLS-RA)culture medium,we suppressed the expression of CD146 in EAhy.926 cells by using lentivirus mediated RNA interference technology.We cultivated and extracted the supernatant of HFLS-RA to prepare conditioned culture medium.And then,we cultivated the transfected EAhy.926 cells in HFLS-RA conditioned medium for 24 h to construct a RA injury model,and performed transcriptome sequencing on it to screen differentially expressed genes(DEGs).Through GO and KEGG pathway analysis,we explored the relevant signaling pathways and biological processes involved in CD146 regulated DEGs.The results showed that compared with the control group cells,EAhy.926 cells with CD146 knockdown in HFLS-RA conditioned medium had 341 DEGs,including 173 upregulated genes and 168 downregulated genes;GO analysis and KEGG pathway analysis revealed that differentially expressed genes were mainly enriched in the Wnt signaling pathway,PPAR signaling pathway,inflammatory mediator regulation of TRP channels,and adipocyte cytokine signaling pathway,and were involved in biological processes such as cell adhesion,cell movement,cell localization,and cysteine biosynthesis process.This study found that at the transcriptome level CD146 may affect EAhy.926 through multiple key signaling pathways and genes under the condition of HFLS-RA conditioned medium.This study provided a theoretical basis for exploring the role and mechanism of CD146 in synovial angiogenesis in rheumatoid arthritis.