LRG1通过CCR1促进类风湿性关节炎滑膜成纤维细胞激活的机制研究

Mechanism of LRG1 promoting synovial fibroblast activation in rheumatoid arthritis through CCR1

目的探讨富亮氨酸重复糖蛋白1(LRG1)对类风湿性关节炎(RA)滑膜成纤维细胞增殖、迁移、侵袭及人脐静脉内皮细胞(HUVECs)血管生成的影响及机制。方法将成纤维样滑膜细胞系MH7A细胞分为6组:对照组(未做任何处理)、肿瘤坏死因子-α(TNF-α)组(20 ng/mL TNF-α处理细胞)、阴性对照干扰小RNA(NC siRNA)组(细胞转染NC siRNA后以TNF-α处理)、LRG1 siRNA组(细胞转染LRG1 siRNA后以TNF-α处理)、LRG1 siRNA+Vector组(细胞转染LRG1 siRNA和pcDNA3.1后以TNF-α处理)和LRG1 siRNA+趋化因子受体1(CCR1)组(细胞转染LRG1 siRNA和pcDNA3.1-CCR1后以TNF-α处理)。采用细胞计数试剂盒8(CCK-8)法检测细胞增殖能力;划痕实验和Transwell侵袭实验检测细胞迁移和侵袭能力;MH7A培养基处理HUVECs,采用血管形成实验检测血管生成能力;实时荧光定量逆转录PCR(qRT-PCR)和Western blot检测LRG1、CCR1 mRNA及蛋白表达水平。免疫共沉淀实验(Co-IP)检测LRG1与CCR1的相互作用。结果与对照组比较,各浓度(5、10、20、40 ng/mL)TNF-α组LRG1 mRNA及蛋白表达水平明显升高(P<0.05),且20 ng/mL为最佳浓度。与NC siRNA组相比,LRG1 siRNA组细胞增殖能力[(137±15)%vs.(228±24)%,P<0.05]、迁移能力明显降低,侵袭细胞数明显减少[(56.0±6.7)个vs.(102.0±7.9)个,P<0.05]。沉默LRG1后HUVECs的血管形成能力明显降低。Co-IP结果显示,LRG1可与CCR1相互作用。沉默LRG1可下调CCR1表达水平(P<0.05),而过表达CCR1可逆转LRG1沉默对MH7A细胞增殖、迁移和侵袭的抑制作用。结论LRG1通过CCR1促进MH7A细胞增殖、迁移、侵袭和HUVECs血管生成,为RA的治疗奠定了理论基础。

Objective To investigate the influence and mechanism of leucine-rich repeat glycoprotein 1(LRG1)on proliferation,migration,invasion of synovial fibroblasts in rheumatoid arthritis(RA)and the angiogenesis of human umbilical vein endothelial cells(HUVECs).Methods Fibrous synovial cells line MH7A were divided into six groups:the control group(without any treatment),tumor necrosis factor-α(TNF-α)group(treated with 20 ng/mL TNF-α),negative control interfering small RNA(NC siRNA)group(treated with TNF-αafter transfection of NC siRNA),LRG1 siRNA group(treated with TNF-αafter transfection of LRG1 siRNA),LRG1 siRNA+Vector group(treated with TNF-αafter transfection of LRG1 siRNA and PCDN3.1)and LRG1 siRNA+chemokine receptor 1(CCR1)group(treated with TNF-αafter transfection of LRG1 siRNA and pcDNA3.1-CCR1).Cell proliferation was detected by CCK-8 assay.Cell migration and invasion were analyzed by scratch assay and Transwell invasion assay.HUVECs were treated with culture of MH7A with different treatment,and cell angiogenesis was analyzed by tube formation assay.qRT-PCR and Western blot were used to detect the mRNA and protein levels of LRG1 and CCR1,respectively.Interaction of LRG1 and CCR1 was detected by co-immunoprecipitation(Co-IP).Results Compared with the control group,the expression levels of LRG1 mRNA and protein were significantly increased in the TNF-αgroups with different concentations(5,10,20,40 ng/mL),and 20 ng/mL was the optimal concentration.Compared with the NC siRNA group,the cell proliferation ability[(137±15)%vs.(228±24)%,P<0.05]and migration ability of the LRG1 siRNA group were significantly reduced,and the number of invading cells was significantly reduced(56.0±6.7 vs.102.0±7.9,P<0.05).After silencing LRG1 the angiogenesis ability of HUVECs cells decreased significantly.The results of Co-IP showed that LRG1 was interacted with CCR1.Silencing of LRG1 downregulated the expression of CCR1(P<0.05).On the other hand,overexpression of CCR1 reversed the inhibitory effects of LRG1 silencing on the proliferation,migration and invasion of MH7A cells.Conclusion LRG1 promotes MH7A cell proliferation,migration,invasion as well as HUVECs angiogenesis through CCR1,which lays a theoretical foundation for the treatment of RA.