ABSTRACT

The participation of long noncoding RNAs (lncRNAs) and microRNAs (miRs) in the progression of rheumatoid arthritis (RA) is a key area of investigation. The current study aimed to investigate the action of lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in fibroblast-like synoviocyte (FLS) proliferation and synovitis in RA. A rat model of RA was established. LncRNA NEAT1 expression in the synovial tissues of patients with RA and FLSs from the RA rat model was determined using RT-qPCR. Next, dual luciferase reporter gene assay was applied to investigate the relationship between miR-129/204 and mitogen-activated protein kinase (MAPK)/extracellular regulated protein kinase (ERK). A putative binding relationship between miR-204 and lncRNA NEAT1 was evaluated by RIP assay, and miR-129 promoter methylation was determined using MSP. After the expression of lncRNA NEAT1, miR-129 or miR-204 was altered in FLSs, the extent of ERK1/2 phosphorylation was assessed. In addition, FLS synovitis and proliferation were determined by ELISA and EdU assay, respectively. In RA rats, lncRNA NEAT1 was silenced and miR-129/miR-204 was overexpressed to explore their roles in vivo. LncRNA NEAT1 was upregulated, while miR-129 and miR-204 were downregulated in RA synovial tissues and FLSs. MAPK1 was target gene of both miR-129 and miR-204. LncRNA NEAT1 bound to miR-204 and promoted miR-129 promoter methylation. Silencing lncRNA NEAT1 or overexpressing miR-129/miR-204 enhanced miR-129/miR-204 expression, but reduced the extent of ERK1/2 phosphorylation, proliferation of FLSs, and synovitis in RA. Collectively, silencing lncRNA NEAT1 promoted miR-129 and miR-204 to inhibit the MAPK/ERK signalling pathway, reducing FLS synovitis in RA.

Abbreviations: ACR: American College of Rheumatology; ELISA: Enzyme-linked immunosorbent assay; ERK: extracellular signal-regulated kinase; FLS: fibroblast-like synoviocyte; GADPH: glyceraldehyde-3-phosphate dehydrogenase; HRP: horseradish peroxidase; IFA: Incomplete Freund’s Adjuvant; lncRNAs: long noncoding RNAs; MSP: Methylation-specific PCR; NC: negative control; NEAT1: nuclear paraspeckle assembly transcript 1; OD: optical density; RA: rheumatoid arthritis; RIPA: Radio Immunoprecipitation Assay; RLU: relative light units; RT-qPCR: reverse transcription quantitative polymerase chain reaction; UTR: untranslated region.

摘要

长链非编码 RNA (lncRNA) 和 microRNA (miR) 在类风湿性关节炎 (RA) 进展中的参与是一个关键的研究领域。本研究旨在探讨 lncRNA 核副啄木鸟组装转录物 1 (NEAT1) 在 RA 成纤维细胞样滑膜细胞 (FLS) 增殖和滑膜炎中的作用。建立大鼠RA模型。使用 RT-qPCR 测定来自 RA 大鼠模型的 RA 和 FLS 患者滑膜组织中 LncRNA NEAT1 的表达。接下来，应用双荧光素酶报告基因测定来研究 miR-129/204 与丝裂原活化蛋白激酶 (MAPK)/细胞外调节蛋白激酶 (ERK) 之间的关系。通过 RIP 测定评估 miR-204 和 lncRNA NEAT1 之间推定的结合关系，并使用 MSP 确定 miR-129 启动子甲基化。在 FLS 中改变 lncRNA NEAT1、miR-129 或 miR-204 的表达后，评估 ERK1/2 磷酸化的程度。此外，分别通过ELISA和EdU测定法测定FLS滑膜炎和增殖。在 RA 大鼠中，lncRNA NEAT1 被沉默，miR-129/miR-204 过表达以探索它们的作用在体内。LncRNA NEAT1 上调，而 miR-129 和 miR-204 在 RA 滑膜组织和 FLS 中下调。MAPK1 是 miR-129 和 miR-204 的靶基因。LncRNA NEAT1 与 miR-204 结合并促进 miR-129 启动子甲基化。沉默 lncRNA NEAT1 或过表达 miR-129/miR-204 可增强 miR-129/miR-204 的表达，但会降低 RA 中 ERK1/2 磷酸化、FLS 增殖和滑膜炎的程度。总的来说，沉默 lncRNA NEAT1 促进 miR-129 和 miR-204 抑制 MAPK/ERK 信号通路，减少 RA 中的 FLS 滑膜炎。

缩写： ACR：美国风湿病学会；ELISA：酶联免疫吸附试验；ERK：细胞外信号调节激酶；FLS：成纤维细胞样滑膜细胞；GADPH：3-磷酸甘油醛脱氢酶；HRP：辣根过氧化物酶；IFA：不完全弗氏佐剂；lncRNAs：长链非编码 RNA；MSP：甲基化特异性 PCR；NC：阴性对照；NEAT1：核副啄木鸟组装转录本 1；OD：光密度；RA：类风湿性关节炎；RIPA：放射免疫沉淀测定；RLU：相对光单位；RT-qPCR：逆转录定量聚合酶链反应；UTR：未翻译区域。