Objectives: Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and joint destruction. Both inflammatory response and oxidative stress contribute to the pathogenesis of RA. Oxidative damage can induce and aggravate the imbalance of immune inflammation and promote cell and tissue damage. In this study, the expression of long non-coding RNA (lncRNA) LINC00638 in peripheral blood of patients with RA damp-heat arthralgia syndrome was observed, and the correlation between LINC00638 and disease activity, immune inflammation and oxidative stress indicator was investigated. Subsequently, the mechanisms for LINC00638 in regulating the inflammatory response and oxidative stress in RA fibroblast-like synoviocyte (FLS) under the condition of overexpression and interference were further explored.
Methods: In this study, 48 RA patients with damp-heat arthralgia syndrome and 27 normal healthy subjects, who came from Department of Rheumatology, First Affiliated Hospital of Anhui University of Chinese Medicine, were included; and they were divided into a RA group and a control group. The expression of LINC00638 in peripheral blood mononuclear cells (PBMC) from the subjects was detected by real-time PCR. Enzyme linked immunosorbent assay (ELISA) was used to detect serum interleukin (IL)-10, IL-17, tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), heme oxygenase 1 (HO-1) and superoxide dismutase 2 (SOD2) expression. Spearman method was used to study the relationship between LINC00638 and erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF), anti-cyclic citrullinated peptide antibody (anti-CCP), and to observe the relation between LINC00638 and the Disease Activity Score of 28 Joint (DAS28), Quantitative Score of Damp Heat Syndrome, Visual Analogue Scale (VAS), Self-rating Anxiety Scale (SAS) and Self-rating Depression Scale (SDS). RA-FLS was induced by RA-PBMC, and the RA in vitro cell experimental model was established. LINC00638 overexpression plasmid and small interfering RNA (siRNA) were constructed and transfected into RA-FLS. The cell experiments were divided into 4 groups: a pcDNA3. 1- control group, a pcDNA3.1-LINC00638 group, a siRNA-control group, and a siRNA-LINC00638 group. The transfection efficiency of overexpression plasmid and siRNA was detected by real-time PCR, the expression of TNF-α and IL-10 was detected by ELISA, and the expression of antioxidant proteins HO-1 and SOD2 was detected by immunofluorescence.
Results: Compared with the control group, the expression of LINC00638 in the RA group was lower (P<0.01). The area under the curve (AUC) of the receiver operating characteristic (ROC) curve of LINC00638 was 0.9271. The DAS28 in RA group was 5.70 (5.40-6.50), the Quantitative Score of Damp-heat Syndrome was 20.0 (17.0-23.0), and the VAS score was 7.0 (6.3-8.0). Compared with the control group, the ESR, CRP, RF, anti-CCP, SAS and SDS scores in the RA group were significantly increased (all P<0.01). Spearman correlation analysis showed that: LINC00638 was negatively correlated with ESR (r=-0.532, P<0.01), CRP (r=-0.367, P<0.05), TNF-α (r=-0.375, P<0.01), MDA (r= -0.295, P<0.05), DAS28 (r=-0.450, P<0.01), and which was positively correlated with SOD2 (r=0.370, P<0.05). After the induction of RA-FLS, the expression level of LINC00638 was significantly decreased (P<0.01), indicating that the stimulation of PBMC could effectively reduce the expression of LINC00638 in RA-FLS, so the experimental model of RA-FLS-induced by PBMC was utilized. Compared with the pcDNA3.1-control group, the expressions of LINC00638, IL-10, SOD2, and HO-1 in the pcDNA3.1-LINC00638 group were significantly increased (all P<0.01), and the expression of TNF-α was decreased (P<0.01). Compared with siRNA-control group, LINC00638, IL-10, SOD2 and HO-1 in the siRNA-LINC00638 group were significantly decreased (all P<0.01), and TNF-α was significantly increased (P<0.01).
Conclusions: LINC00638 is down-regulated in the peripheral blood of RA patients with damp-heat arthralgia syndrome, which is correlated with disease activity, immune inflammation and oxidative stress. Overexpression of LINC00638 can down-regulate pro-inflammatory factors, up-regulate anti-inflammatory factors, and increase antioxidant enzyme activity, thereby improving inflammation and oxidative stress in RA. LINC00638 is the differential lncRNA obtained by the research group's previous high-throughput sequencing of the whole transcriptome of peripheral blood PBMCs in RA patients and validation of clinical samples. In order to deepen the molecular biology research of this gene, the microRNA and mRNA targeted by LINC00638 can be further studied from the perspective of competing endogenous RNAs.
目的: 类风湿关节炎(rheumatoid arthritis，RA)是以慢性炎症和关节破坏为主要特征的自身免疫性疾病。炎症反应、氧化应激是RA发病的重要环节，氧化损伤能够诱发和加重免疫炎症失衡，促进细胞和组织损伤。本研究观察长链非编码RNA (long non-coding RNA，lncRNA) LINC00638在RA湿热痹阻证患者外周血中的表达，旨在研究LINC00638与疾病活动度、免疫炎症、氧化应激指标的相关性，探讨LINC00638在过表达和干扰情况下调控RA滑膜样成纤维细胞(fibroblast-like synoviocyte，FLS)的炎症反应与氧化应激的机制。方法: 纳入安徽中医药大学第一附属医院风湿科48例RA湿热痹阻证患者和27例正常健康者，将其分为RA组和对照组。采用real-time PCR检测受试者外周血单个核细胞(peripheral blood mononuclear cell，PBMC)中LINC00638的表达。采用酶联免疫吸附法(enzyme linked immunosorbent assay，ELISA)检测血清中白介素(interleukin，IL)-10、IL-17、肿瘤坏死因子-α(tumor necrosis factor -α，TNF-α)、丙二醛(malondialdehyde，MDA)、血红素氧合酶1(heme oxygenase-1，HO-1)、超氧化物歧化酶2(superoxide dismutase 2，SOD2)的表达。采用Spearman方法研究RA患者LINC00638与红细胞沉降率(erythrocyte sedimentation rate，ESR)、C反应蛋白(C-reactive protein，CRP)、类风湿因子(rheumatoid factor，RF)、抗环瓜氨酸肽抗体(anti-cyclic citrullinated peptide antibody，anti-CCP)的相关性，并观察其与28处关节疾病活动评分(Disease Activity Scores for 28 Joints，DAS28)、RA湿热证候量化积分、视觉模拟评分(Visual Analogue Scale，VAS)、焦虑自评量表(Self-rating Anxiety Scale，SAS)、抑郁自评量表(Self-rating Depression Scale，SDS)评分的关系。以RA-PBMC诱导RA-FLS，建立RA体外细胞实验模型，构建LINC00638过表达质粒和小干扰RNA(small interfering RNA，siRNA)，并将其转染至RA-FLS中。细胞实验分为4组：pcDNA3.1-对照组、pcDNA3.1-LINC00638组、siRNA-对照组、siRNA-LINC00638组。采用real-time PCR检测过表达质粒和siRNA转染效率，ELISA法检测各组TNF-α、IL-10表达，免疫荧光法检测各组抗氧化蛋白HO-1、SOD2的表达。结果: 与对照组相比，LINC00638在RA组外周血中呈低表达(P<0.01)，LINC00638的受试者工作特征(receiver operating characteristic，ROC)曲线下面积(area under curve，AUC)为0.9271。RA组DAS28评分为5.70(5.40~6.50)，RA湿热证候量化积分为20.00(17.00~23.00)，VAS评分为7.00(6.30~8.00)。与对照组相比，RA组ESR、CRP、RF、anti-CCP、SAS评分、SDS评分均显著升高(均P<0.01)。Spearman相关性分析结果显示：RA湿热痹阻证患者LINC00638与ESR(r=-0.532，P<0.01)、CRP(r=-0.367，P<0.05)、TNF-α(r= -0.375，P<0.01)、MDA(r=-0.295，P<0.05)、DAS28 (r=-0.450，P<0.01)均呈负相关，与SOD2呈正相关(r=0.370， P<0.05)。RA-FLS经RA-PBMC诱导后，LINC00638表达水平显著降低(P<0.01)，表明PBMC的刺激能够有效降低RA-FLS的LINC00638表达，故可采用PBMC诱导的RA-FLS实验模型。与pcDNA3.1-对照组相比，pcDNA3.1-LINC00638组LINC00638、IL-10、SOD2、HO-1表达均显著升高(均P<0.01)，TNF-α的表达降低(P<0.01)；与siRNA对照组相比，siRNA-LINC00638组LINC00638、IL-10、SOD2、HO-1均显著降低(均P<0.01)，TNF-α显著升高(P<0.01)。结论: LINC00638在RA湿热痹阻证患者外周血中呈低表达，与疾病活动度、免疫炎症、氧化应激指标存在相关性。过表达LINC00638能够降下调促炎因子，上调抑炎因子，提高抗氧化酶活性，从而可改善RA炎症与氧化应激。LINC00638是本课题组前期对RA患者外周血PBMC进行全转录组高通量测序，以及临床样本验证所得到的差异lncRNA。为深化该基因在分子生物学中研究，可从竞争性内源RNA角度进一步研究LINC00638靶向的微小RNA与mRNA。.
Keywords: LINC00638; damp-heat arthralgia syndrome; inflammation; oxidative stress; rheumatoid arthritis.