MicroRNA-101-3p inhibits fibroblast-like synoviocyte proliferation and inflammation in rheumatoid arthritis by targeting PTGS2

Abstract

Objective: Rheumatoid arthritis (RA) is the most frequently occurring inflammatory arthritis. The present study was performed to characterize the role of microRNA-101-3p (miR-101-3p) and prostaglandin-endoperoxide synthase 2 (PTGS2) in inflammation and biological activities of fibroblast-like synoviocytes (FLSs) in RA.

Methods: Initially, miR-101-3p and PTGS2 expression in RA tissues of RA patients and RA rats was detected by qRT-PCR and Western blot analysis. Rat model of type II collagen-induced arthritis (CIA) was adopted to simulate RA, followed by injection of miR-101-3p mimics or siRNA against PTGS2. Next, the apoptosis in synovial tissue and the levels of tumor necrosis factor (TNF)-α, IL-1β and IL-6 were identified. Subsequently, FLSs in RA (RA-FLSs) were isolated, after which in vitro experiments were conducted to analyze cell proliferation, apoptosis, migration and invasion upon treatment of up-regulated miR-101-3p and silenced PTGS2. Furthermore, the relationship of miR-101-3p and PTGS2 was determined by bioinformatics prediction and luciferase activity assay.

Results: We identified poorly expressed miR-101-3p and highly expressed PTGS2 in synovial tissues of RA patients and RA rats, which showed reduced synoviocyte apoptosis and enhanced inflammation. In response to miR-101-3p mimics and si-PTGS2, the RA-FLSs were observed with attenuated cell proliferation, migration and invasion, corresponding to promoted apoptosis. Down-regulation of PTGS2 could rescue the effect of inhibited miR-101-3p in synovial injury and phenotypic changes of FLS in RA rats. Notably, miR-101-3p was found to negatively regulate PTGS2.

Conclusion: Taken together, miR-101-3p reduces the joint swelling and arthritis index in RA rats by down-regulating PTGS2, as evidenced by inhibited FLS proliferation and inflammation.

Keywords: Fibroblast-like synoviocytes; Prostaglandin-endoperoxide synthase 2; Rheumatoid arthritis; Synovial injury; microRNA-101-3p.

Figure 1. Expression levels of miR-101-3p and PTGS2 in synovial tissue of rats in each group

(A) qRT-PCR detection of miR-101-3p and PTGS2 expression in synovial tissue of patients with RA. (B) Western blot analysis detection of PTGS2 protein expression in synovial tissue of patients with RA. (C) Expression levels of miR-101-3p and PTGS2 in synovial tissue of RA rats. (D) Western blot analysis detection of PTGS2 protein expression in synovial tissue of RA rats. (E) Expression levels of miR-101-3p and PTGS2 in synovial tissue of RA rats with interference of miR-101-3p and PTGS2 expression. (F) PTGS2 protein expression in synovial tissue of RA rats with interference of miR-101-3p and PTGS2 expression. The data analysis between the two groups was compared with the t test. The data analysis among multiple groups was performed using one-way ANOVA, followed by pairwise comparison using LSD-t. *P<0.05 vs. the normal tissues. ^aP<0.05 vs. the normal group; ^bP<0.05 vs. the mimic-NC group; ^cP<0.05 vs. the sh-NC group; ^dP<0.05 vs. the miR-101-3p inhibitors + sh-NC group.

Figure 2. Quantitation of arthritis scores of rats in each group

The data analysis was performed using one-way ANOVA, followed by pairwise comparison using LSD-t. ^aP<0.05 vs. the normal group; ^bP<0.05 vs. the mimic-NC group; ^cP<0.05 vs. the sh-NC group; ^dP<0.05 vs. the miR-101-3p inhibitors + sh-NC group.

Figure 3. Pathological observation of synovial tissues and apoptosis of synoviocytes in each group

(A) HE staining was used to observe the pathological condition of synovial tissue in each group (×400). (B) TUNEL staining was used to detect the apoptosis of rat synoviocytes (×200). (C) The apoptosis cells in each group. (D) Western blot assay was used to detect the expression of apoptosis-related proteins in the synovial tissue. The data analysis was performed using one-way ANOVA, followed by pairwise comparison using LSD-t. ^aP<0.05 vs. the normal group; ^bP<0.05 vs. the mimic-NC group; ^cP<0.05 vs. the sh-NC group; ^dP<0.05 vs. the miR-101-3p inhibitors + sh-NC group.

Figure 4. Quantitation of the mRNA expression of inflammatory factors in synovial tissue and their serum levels of rats in each group

(A) qRT-PCR was used to detect the mRNA expression of inflammatory factors in rat synovial tissue. (B) ELISA was used to detect the serum levels of inflammatory factors in rats. n=5. The data analysis was performed using one-way ANOVA, followed by pairwise comparison using LSD-t. ^aP<0.05 vs. the normal group; ^bP<0.05 vs. the mimic-NC group; ^cP<0.05 vs. the sh-NC group; ^dP<0.05 vs. the miR-101-3p inhibitors + sh-NC group.

Figure 5. Morphological observation and immunocytochemical identification of FLSs

(A) Morphology of third-passage FLSs of RA (400×). (B) Vimentin immunocytochemical staining of FLSs (200×).

Figure 6. MTT assay verifies the effect of miR-101-3p and PTGS2 on the proliferation of RA-FLSs after transfection

The data analysis was performed using one-way ANOVA, followed by pairwise comparison using LSD-t. The experiment was repeated three times. ^aP<0.05 vs. the mimic-NC group; ^bP<0.05 vs. the sh-NC group; ^cP<0.05 vs. the miR-101-3p inhibitors + sh-NC group.

Figure 7. Annexin V-FITC/PI double staining and Hoechst 33258 staining of apoptosis detection in RA-FLSs after transfection

(A) Flow cytometric detection of RA-FLS apoptosis in each group. (B) Hoechst 33258 staining for RA-FLS apoptosis in each group. The data analysis was performed using one-way ANOVA, followed by pairwise comparison using LSD-t. The experiment was repeated three times. ^aP<0.05 vs. the mimic-NC group; ^bP<0.05 vs. the sh-NC group; ^cP<0.05 vs. the miR-101-3p inhibitors + sh-NC group.

Figure 8. Scratch test and Transwell invasion assay indicate the roles of miR-101-3p and PTGS2 in cell migration and invasion ability of RA-FLSs after transfection

(A,B) Representative images (×100) and quantitation of scratch healing rate of each group detected by scratch test. (C,D) Representative images (×200) and quantitation of the number of invasive cells of each group detected by Transwell invasion assay. The data analysis was performed using one-way ANOVA, followed by pairwise comparison using LSD-t. The experiment was repeated three times. ^aP<0.05 vs. the mimic-NC group; ^bP<0.05 vs. the sh-NC group; ^cP<0.05 vs. the miR-101-3p inhibitors + sh-NC group.

Figure 9. Targeting relationship of miR-101-3p and PTGS2 determined based on bioinformatics prediction and luciferase activity assay

(A) qRT-PCR was used to detect the expression of miR-101-3p and PTGS2 in each group. (B) Western blot assay was used to detect the protein expression of PTGS2 in each group. (C) Binding sites between miR-101-3p and PTGS2 were predicted by bioinformatics analysis. (D) Dual luciferase reporter assay analysis. The data analysis was performed using one-way ANOVA, followed by pairwise comparison using LSD-t. The experiment was repeated three times. ^aP<0.05 vs. the mimic-NC group; ^bP<0.05 vs. the sh-NC group; ^cP<0.05 vs. the miR-101-3p inhibitors + sh-NC group.