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). 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 ~2,000-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 2 C-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 4 F-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.