Expression of LINC00638 in rheumatoid arthritis patients with damp-heat obstruction syndrome and the regulatory mechanisms for inflammation and oxidative stress

Abstract

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.

Keywords: LINC00638; damp-heat arthralgia syndrome; inflammation; oxidative stress; rheumatoid arthritis.

图1

LINC00638在RA组PBMC中的表达及ROC曲线分析 Figure 1 Expression of LINC00638 in PBMC and ROC curve analysis in the RA group A: Low expression of LINC00638 in PBMC in the RA group by real-time PCR; B: Diagnostic utility of LINC00638 by ROC curve. **P<0.01 vs the control group.

图2

RA和对照组血清胞因子及氧化应激指标表达的比较 Figure 2 Comparison of expression of serum cytokines and oxidative stress indicators between the RA group and the control group A: IL-10; B: IL-17; C: TNF-α; D: MDA; E: HO-1; F: SOD2. **P<0.01 vs the control group.

图3

LINC00638与RA组临床指标的相关性分析 Figure 3 Correlation analysis between LINC00638 and clinical indexes in the RA group A: Correlation analysis between LINC00638 and ESR; B: Correlation analysis between LINC00638 and CRP; C: Correlation analysis between LINC00638 and TNF-α; D: Correlation analysis between LINC00638 and MDA; E: Correlation analysis between LINC00638 and SOD2; F: Correlation analysis between LINC00638 and DAS28.

图4

LINC00638过表达质粒与小干扰RNA的转染效率 Figure 4 Transfection efficiency of LINC00638 overexpressed plasmid and siRNA A: LINC00638 expression in RA-FLS stimulated by RA-PBMC. **P<0.01 vs the RA-FLS. B: Detection efficiency of LINC00638 overexpressed plasmid and siRNA by real-time PCR. **P<0.01 vs the pcDNA3.1-control group; ††P<0.01 vs the siRNA-control group.

图5

LINC00638对RA-FLS中TNF-α、IL-10表达的影响 Figure 5 Effect of LINC00638 on the expression of TNF-α and IL-10 in RA-FLS A: Expression of TNF-α by ELISA; B: Expression of IL-10 by ELISA. **P<0.01 vs the pcDNA3.1-control group; ††P<0.01 vs the siRNA-control group.

图6

LINC00638对RA-FLS的HO-1和SOD2蛋白表达的影响 Figure 6 Effect of LINC00638 on HO-1 and SOD2 protein expression in RA-FLS A: Expression of HO-1 protein by immunofluorescence assay; B: Expression of SOD2 protein by immunofluorescence assay. Merge: Dyeing superposition. **P<0.01 vs the pcDNA3.1-control group; ††P<0.01 vs the siRNA-control group.