miR-29a对类风湿关节炎成纤维样滑膜细胞增殖和凋亡的影响

基础医学与临床 ›› 2020, Vol. 40 ›› Issue (1): 9-15.

miR-29a对类风湿关节炎成纤维样滑膜细胞增殖和凋亡的影响

郭雄飞^*, 王挺, 汤立新

河南省南阳市中心医院郑州大学附属南阳医院骨科, 河南南阳 473000

收稿日期:2019-04-03 修回日期:2019-07-08 出版日期:2020-01-05 发布日期:2019-12-27
通讯作者: ^*3515346698@qq.com
基金资助:
2018年度河南省重点研发与推广专项(182102310466)

Effect of miR-29a on proliferation and apoptosis of fibroblast-like synoviocyte in rheumatoid arthritis

GUO Xiong-fei^*, WANG Ting, TANG Li-xin

Department of Orthopaedics, Nanyang Central Hospital, the Affiliated Nanyang Hospital, Zhengzhou University, Nanyang 473000, China

Received:2019-04-03 Revised:2019-07-08 Online:2020-01-05 Published:2019-12-27
Contact: ^*3515346698@qq.com

摘要/Abstract

摘要： 目的探讨miR-29a对类风湿关节炎(RA)成纤维样滑膜细胞(FLS)活力和凋亡的影响。方法分离培养健康人FLS(n-FLS)和RA患者FLS(RA-FLS);Western blot检测n-FLS和RA-FLS中标志物CHI3L1蛋白表达;将miR-29a mimics、si-NFAT5及miR-29a+NFAT5转染RA-FLSs,转染48 h后,RT-qPCR检测miR-29a和NFAT5 mRNA表达;MTT法及流式细胞计量术分别检测各组细胞活力和凋亡率;Western blot检测p38、p-p38、cleaved-caspase3蛋白表达;通过双荧光报告基因检测系统检测过表达或抑制miR-29a后NFAT5表达,验证miR-29a和NFAT5的靶向关系。结果与n-FLS比较,RA-FLS中CHI3L1蛋白表达明显升高(P＜0.05);过表达miR-29a或抑制NFAT5后,RA-FLS细胞活力明显降低,凋亡率明显升高,p-p38表达明显降低,cleaved-caspase3表达明显升高(P＜0.05)。miR-29a与野生型NFAT5 3′UTR共转染组RA-FLS的荧光素酶活性明显降低,而anti-miR-29a与野生型NFAT5 3′UTR共转染组荧光素酶活性明显升高(P＜0.05);过表达miR-29a可明显下调RA-FLS中NFAT5表达,抑制miR-29a表达可明显上调NFAT5表达(P＜0.05);过表达NFAT5可减弱过表达miR-29a对RA-FLS活力和凋亡的影响(P＜0.05)。结论 miR-29a可通过靶向NFAT5下调p38MAPK信号通路抑制RA-FLS活力及诱导细胞凋亡。

关键词: 类风湿关节炎, 滑膜样细胞, miR-29a靶基因, 活化T细胞核因子5, p38MAPK信号通路

Abstract: Objective To investigate the effect of miR-29a on the activity and apoptosis of fibroblast-like synoviocyte(FLS) in rheumatoid arthritis(RA). Methods The normal human FLS (n-FLS) and RA patients FLS (RA-FLS) were isolated and cultured,and the expression level of CHI3L1 protein was detected by Western blot. miR-29a mimics, si-NFAT5 and miR- 29a+NFAT5 were transfected into RA-FLS for 48 h. Then,the expression levels of miR-29a and NFAT5 mRNA were detected by RT-qPCR, the cell viability and apoptosis were detected by MTT assay and flow cytometry,respectively and the expression of p38, p-p38, cleaved-caspase3 proteins were detected by Western blot. Targeting relationship between miR-29a and NFAT5 was validated by dual fluorescent reporter gene detection system. Results Compared with n-FLS, the expression level of CHI3L1 protein in RA-FLS was increased significantly (P＜0.05). After over-expression of miR-29a or inhibition of NFAT5, the cell viability decreased, the apoptosis rate increased, the expression of p-p38 decreased, and the expression of cleaved-caspase3 increased significantly(P＜0.05). Luciferase activity of RA-FLS was significantly decreased in miR-29a and wild-type NFAT5 3′UTR co-transfection group, but increased in anti-miR-29a and wild-type NFAT5 3′UTR co-transfection group (P＜0.05). Over-expression of miR-29a significantly decreased the expression of NFAT5, and inhibition of miR-29a significantly increased the expression of NFAT5 (P＜0.05).Over-expression of NFAT5 can weaken the effect of over-expression of miR-29a on RA-FLS activity and apoptosis (P＜0.05). Conclusions miR-29a can inhibit the activity of RA-FLS and induce its apoptosis by targeting NFAT5 to down-regulate p38 MAPK signaling pathway.

Key words: rheumatoid arthritis(RA), fibroblast-like synoviocyte(FLS), miR-29a target gene, nuclear factor of activated T cells 5(NFAT5), p38MAPK signaling pathway

中图分类号:

R593.22

郭雄飞, 王挺, 汤立新. miR-29a对类风湿关节炎成纤维样滑膜细胞增殖和凋亡的影响[J]. 基础医学与临床, 2020, 40(1): 9-15.

GUO Xiong-fei, WANG Ting, TANG Li-xin. Effect of miR-29a on proliferation and apoptosis of fibroblast-like synoviocyte in rheumatoid arthritis[J]. Basic & Clinical Medicine, 2020, 40(1): 9-15.

参考文献

[1]McInnes IB, Schett G. Pathogenetic insights from the treatment of rheumatoid arthritis[J]. The Lancet, 2017, 389: 2328-2337.
[2]Lebre MC, Vieira PL, Tang MW, et al. Synovial IL-21/TNF-producing CD4⁺ T cells induce joint destruction in rheumatoid arthritis by inducing matrix metalloproteinase production by fibroblast-like synoviocytes[J]. J Leukoc Biol, 2017, 101: 775-783.
[3]Li S, Jin Z, Lu X. MicroRNA-192 suppresses cell proliferation and induces apoptosis in human rheumatoid arth-ritis fibroblast-like synoviocytes by downregulating caveo-lin 1[J]. Mol Cell Biochem, 2017, 432: 123-130.
[4]Liu J, Fei D, Xing J, et al. MicroRNA-29a inhibits proliferation and induces apoptosis in rheumatoid arthritis fibroblast-like synoviocytes by repressing STAT3[J]. Biomed Pharmacother, 2017, 96:173-181.
[5]Yoon HJ, You S, Yoo SA, et al. NF-AT5 is a critical regulator of inflammatory arthritis[J]. Arthritis Rheum, 2011, 63:1843-1852.
[6]Choi S, You S, Kim D, et al. Transcription factor NFAT5 promotes macrophage survival in rheumatoid arthritis[J]. J Clin Invest, 2017, 127:954-969.
[7]Singh JA, Saag KG, Bridges Jr SL, et al. 2015 American College of Rheumatology guideline for the treatment of rheumatoid arthritis[J]. Arthritis Care Res(Hoboken), 2016, 68: 1-26.
[8]Mallinson DJ, Dunbar DR, Ridha S, et al. Identification of potential plasma microRNA stratification biomarkers for response to allogeneic adipose-derived mesenchymal stem cells in rheumatoid arthritis[J]. Stem Cells Transl Med, 2017, 6: 1202-1206.
[9]Ko JY, Lee MS, Lian WS, et al. MicroRNA-29a counteracts synovitis in knee osteoarthritis pathogenesis by targeting VEGF[J]. Sci Rep, 2017, 7:3584-3594.
[10]Böger CA, Gorski M, McMahon GM, et al. NFAT5 and SLC4A10 loci associate with plasma osmolality[J]. J Am Soc Nephrol, 2017, 28: 2311-2321.
[11]Patnaik A, Haluska P, Tolcher AW, et al. A first-in-human phase i study of the oral p38 MAPK inhibitor, ralimetinib (LY2228820 Dimesylate), in patients with advanced cancer[J]. Clin Cancer Res, 2016, 22: 1095-1102.
[12]Kanashiro A, Franchin M, Bassi GS, et al. Inhibition of spinal p38 MAPK prevents articular neutrophil infiltration in experimental arthritis via sympathetic activation[J]. Fundam Clin Pharmacol, 2018, 32: 155-162.
[13]Liang L, Huang M, Xiao Y, et al. Inhibitory effects of niclosamide on inflammation and migration of fibroblast-like synoviocytes from patients with rheumatoid arthritis[J]. Inflamm Res, 2015, 64: 225-233.
[14]Zhai Y, Wu B, Li J, et al. CD147 promotes IKK/IκB/NF-κB pathway to resist TNF-induced apoptosis in rheumatoid arthritis synovial fibroblasts[J]. J Mol Med(Berl), 2016, 94: 71-82.