miR-431-5p was downregulated in RA
qRT-PCR showed a downregulation of miR-431-5p in synovial tissues from patients with RA as compared to that in the healthy cohort (p = 0.0007, Fig. 1a). Furthermore, we detected miR-431-5p levels in immortalized cell lines and HFLS and HFLS-RA cells. Accordingly, miR-431-5p was downregulated in HFLS-RA cells compared to that in HFLS cells (p < 0.0001, Fig. 1b). Moreover, miR-431-5p was reduced in HFLS-RA cells with TNF-α treatment compared with that without TNF-α treatment (p = 0.001, Fig. 1c), suggesting that dysregulated miR-431-5p might be involved in the development of RA.
Overexpression of miR-431-5p suppressed cell proliferation in RA FLSs
To elucidate the effects of miR-431-5p on cell proliferation in RA FLSs, we used the miR-431-5p mimics and inhibitor in HFLS-RA cells. The transfected HFLS-RA cells showed a ~ 2000-fold enhancement in miR-431-5p levels (p < 0.0001, Fig. 2a); HFLS-RA cells transfected with the inhibitor showed > 2-fold reduction in the levels of miR-431-5p (p < 0.0001). The NC samples showed no difference in miR-431-5p levels.
CCK-8 assays showed decreased proliferation in miR-431-5p-overepxressing HFLS-RA cells (p < 0.05, Fig. 2b). However, miR-431-5p inhibition significantly increased cell proliferation (p < 0.05, Fig. 2b). EdU staining assays were consistent with CCK-8 assay; miRNA mimics-transfected cells showed a decrease in the number of EdU-positive cells (p < 0.0001, Fig. 2c, d), while inhibition of miR-431-5p increased the number of EdU-positive cells compared to that of the control subsets (p < 0.0001, Fig. 2c, d). This suggests that upregulation of miR-431-5p suppresses HFLS-RA cell proliferation.
Overexpression of miR-431-5p induced apoptosis and suppressed G0/G1-to-S phase transition in RA FLSs
We used flow cytometry to understand the role of miR-431-5p on apoptosis and cell cycle progression. As shown in Fig. 3a, miR-431-5p overexpression significantly enhanced apoptosis in HFLS-RA cells (p = 0.0004, Fig. 3d), particularly during the early phase of apoptosis (p < 0.05, Fig. 3b). Inhibiting miR-431-5p expression suppressed early and end-phase apoptosis in HFLS-RA cells (p = 0.0201 and p < 0.0001, respectively). However, apoptotic ratios showed no difference among five groups in the late phase of apoptosis in HFLS-RA cells.
Further, we explored the function of miR-431-5p on cell cycle progression in HFLS-RA cells. Flow cytometry showed that the ratio of G0/G1 phase HFLS-RA cells was significantly higher in cells transfected with the miR-431-5p mimics (p = 0.0253, Fig. 3e, f), while the ratio of G0/G1 phase HFLS-RA cells was lower in cells depleted of miR-431-5p as compared to that in their respective control subsets (p < 0.05). Thus, miR-431-5p may inhibit G0/G1-to-S phase transition in HFLS-RA cells. Conclusively, overexpression of miR-431-5p might suppress cell proliferation through inducing apoptosis and the G0/G1-to-S phase transition in RA FLSs.
miR-431-5p directly bound XIAP in RA FLSs
The putative binding between miR-431-5p and XIAP was predicted by TargetScan (Fig. 4a). As shown in Fig. 4b, the miR-431-5p mimics reduced luciferase activity when co-transfected with the construct containing the WT 3′ UTR of XIAP (p = 0.0005). However, we observed no difference in luciferase activity in cells co-transfected with the construct containing XIAP 3′ UTR mutant, indicating binding between miR-431-5p and XIAP in HFLS-RA cells.
qRT-PCR and western blotting showed that miR-431-5p mimics significantly reduced the mRNA and protein levels of XIAP (p = 0.018 and p = 0.0069, respectively, Fig. 4c–e), while XIAP levels were induced in HFLS-RA cells after transfection with the inhibitor (p = 0.0108 and p = 0.0007, respectively). These results confirmed the interaction between miR-431-5p and XIAP in RA FLSs.
To further explore the miR-431-5p/XIAP signaling in RA, we determined the levels of XIAP in synovial tissues and cells. As shown in Fig. 4f–h, the mRNA and protein levels of XIAP were higher in synovial tissues of patients with RA as compared to that in the healthy cohort (p = 0.0069 and p = 0.0004, respectively). Consistently, the mRNA and protein levels of XIAP were upregulated in HFLS-RA cells as compared to that in HFLS cells (p = 0.0006 and p = 0.0001, respectively). Taken together, miR-431-5p may contribute to the development of RA by regulating XIAP.
Our previous studies have shown another miRNA, miR-410-3p, regulates cell proliferation, apoptosis, and cell cycle by directly targeting YinYang 1 in RA FLSs [16]. Since miR-431-5p shared overlapping effects with miR-410-3p in RA FLSs, we explored whether miR-431-5p and miR-410-3p also share similar mechanisms. As shown in Supplementary Fig 1 A-C, there were no significant differences of YY1 levels in HFLS-RA cells after transfection with miR-431-5p mimics, inhibitor and their respective NCs (all p > 0.05). However, XIAP levels were significantly inhibited in HFLS-RA cells after transfection with miR-410-3p mimics (p = 0.0006 and p = 0.0001, respectively, Supplementary Fig 2 A-C), suggesting that miR-431-5p and miR-410-3p might exert similar effects in RA FLSs through overlapping mechanisms.
miR-431-5p regulated cell proliferation, apoptosis, and cell cycle progression via XIAP in RA FLSs
To understand the mechanism employed by miR-431-5p in regulating cell proliferation, apoptosis, and cell cycle progression in RA FLSs, we manipulated the expression of XIAP using siRNAs in HFLS-RA cells. As shown in Fig. 5a–c, siRNAs against XIAP reduced the mRNA and protein levels of XIAP in HFLS-RA (p < 0.05, p < 0.05, and p > 0.05, respectively). Since the siRNAs showed varied efficiency, we selected siRNA#1 and siRNA#2 for our subsequent functional assays.
CCK-8 assay showed that promotion of cell proliferation mediated by miR-431-5p inhibition was partially restored by XIAP silencing (all p < 0.05, Fig. 5d), particularly at 48 h, 72 h, and 96 h. Consistently, EdU staining indicated that the population of EdU-positive cells was lower in the cells co-transfected with the miRNA inhibitor and siRNAs against XIAP as compared to that in the cells transfected with the miRNA inhibitor (both p < 0.0001, Fig. 5e, f). Furthermore, inhibition of apoptosis induced by miR-431-5p inhibitor was restored by XIAP silencing (p < 0.0001 and p = 0.0001, respectively). Moreover, flow cytometry showed that the ratio of G0/G1 phase HFLS-RA cells was higher in cells co-transfected with the miRNA inhibitor and siRNAs against XIAP as compared to that in cells only transfected with the inhibitor (p = 0.0084 and p = 0.0068, respectively). This suggests that the increase in G0/G1-to-S phase transition induced by the miR-431-5p inhibitor was partially reduced by XIAP siRNAs. Thus, miR-431-5p suppressed cell proliferation and G0/G1-to-S phase transition and promoted apoptosis by targeting XIAP in RA FLSs.