背景:环状RNA(circularRNAs,circRNAs)是类风湿性关节炎(RA)发病和进展的重要调节因子。我们的目的是探讨circ_0000396在RA进展中的作用和支持机制。
方法:从陕西中医药大学附属医院招募RA患者(n=39)和健康志愿者(n=33)。通过逆转录-定量聚合酶链反应分析Circ_0000396、microRNA-574-5p(miR-574-5p)和R-spondin1(RSPO1)RNA水平。通过3-(4,5-二甲基-噻唑-2-基)-2,5-二苯基四唑溴化物(MTT)测定法分析细胞增殖,集落形成试验,和5-乙炔基-2'-脱氧尿苷(EDU)测定。通过流式细胞术评估细胞凋亡。增殖细胞核抗原(PCNA)的蛋白表达水平,细胞周期蛋白D1,细胞周期蛋白E1,BCL2相关×蛋白(Bax),B细胞淋巴瘤-2(Bcl2),白细胞介素-1β(IL-1β),Westernblot检测肿瘤坏死因子-α(TNF-α)和RSPO1。采用酶联免疫吸附试验(ELISA)分析IL-1β和TNF-α等促炎细胞因子的分泌情况。miR-574-5p与circ_0000396或RSPO1之间的相互作用通过双荧光素酶报告基因测定和RNA下拉测定来确认。
结果:与健康对照组相比,RA患者中Circ_0000396表达显著下调。Circ_0000396过表达抑制RA滑膜成纤维细胞(RASFs)的增殖和炎症反应,并引发凋亡,伴随着PCNA的减少,CyclinD1、CyclinE1、Bcl2、IL-1β和TNF-α蛋白表达增加而Bax蛋白表达增加。Circ_0000396充当miR-574-5p的分子海绵,通过引入miR-574-5p模拟物,circ_0000396过表达介导的对RASF功能障碍的保护作用在很大程度上被逆转。miR-574-5p与RSPO1的3'非翻译区(3'UTR)相互作用,miR-574-5p负调控RASF中的RSPO1表达。Circ_0000396可以通过在RASF中复制miR-574-5p来上调RSPO1的表达。RSPO1干扰在很大程度上推翻了RASF中circ_0000396过表达介导的作用。
结论:Circ_0000396通过介导miR-574-5p/RSPO1轴抑制RASF的增殖和炎症反应并诱导其凋亡,这为RA治疗提供了新的潜在靶点。
BACKGROUND: Circular RNAs (circRNAs) are important regulators on the onset and progression of rheumatoid arthritis (RA). Our purpose is to explore the role and underpin mechanism of circ_0000396 in RA progression.
METHODS: RA patients (n = 39) and healthy volunteers (n = 33) were recruited from the Affiliated Hospital of Shaanxi University of Chinese Medicine for the present work. Circ_0000396, microRNA-574-5p (miR-574-5p) and R-spondin 1 (
RSPO1) RNA levels were analyzed by reverse transcription-quantitative polymerase chain reaction. Cell proliferation was analyzed by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, and 5-ethynyl-2\'-deoxyuridine (EDU) assay. Cell apoptosis was assessed by flow cytometry. Protein expression levels of proliferating cell nuclear antigen (PCNA), Cyclin D1, Cyclin E1, BCL2-associated × protein (Bax), B-cell lymphoma-2 (Bcl2), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and RSPO1 were detected by western blot assay. Enzyme-linked immunosorbent assay (ELISA) was conducted to analyze the secretion of pro-inflammatory cytokines including IL-1β and TNF-α. The interaction between miR-574-5p and circ_0000396 or
RSPO1 was confirmed by dual-luciferase reporter assay and RNA-pull down assay.
RESULTS: Circ_0000396 expression was notably down-regulated in RA patients compared with healthy controls. Circ_0000396 overexpression suppressed the proliferation and inflammatory response and triggered the apoptosis of RA synovial fibroblasts (RASFs), accompanied by decreases in PCNA, Cyclin D1, Cyclin E1, Bcl2, IL-1β and TNF-α protein expression and an increase in Bax protein expression. Circ_0000396 acted as a molecular sponge for miR-574-5p, and circ_0000396 overexpression-mediated protective effects on RASFs dysfunction were largely reversed by the introduction of miR-574-5p mimics. miR-574-5p interacted with the 3\' untranslated region (3\'UTR) of RSPO1, and miR-574-5p negatively regulated
RSPO1 expression in RASFs. Circ_0000396 could up-regulate the expression of
RSPO1 by sponging miR-574-5p in RASFs.
RSPO1 interference largely overturned circ_0000396 overexpression-mediated effects in RASFs.
CONCLUSIONS: Circ_0000396 restrained the proliferation and inflammation and induced the apoptosis of RASFs by mediating miR-574-5p/RSPO1 axis, which provided novel potential targets for RA treatment.