Hypoxia-induced miR-191-C/EBPβ signaling regulates cell proliferation and apoptosis of fibroblast-like synoviocytes from patients with rheumatoid arthritis

Abstract

Background: Hypoxia plays an important role in the proliferation of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS), leading to pathology of RA. This study was conducted to evaluate hypoxia-induced microRNAs (hypoxamiR) in RA-FLS and its role in the function of RA-FLS.

Methods: RA-FLS were cultured under normoxia (21% O₂) or hypoxia (3% O₂) condition, followed by a microRNA (miRNA) array analysis. The upregulation of miR-191 by hypoxia was confirmed in RA-FLS and FLS from osteoarthritis (OA) patients by quantitative real-time polymerase chain reaction (RT-PCR). Transfection of miR-191 mimic and inhibitor was used to investigate the function of miR-191 in RA-FLS. The functional targets of miR-191 were predicted by bioinfomatics and then validated by reporter gene assay.

Results: A subset of miRNAs was identified to be induced by hypoxia including miR-191. The upregulation of miR-191 was found to be specific in hypoxic RA-FLS, compared to hypoxic OA-FLS. We observed that miR-191 in RA-FLS increased cellular proliferation via promoting G₁/S transition of the cell cycle and suppressed cell apoptosis induced by cell starvation. Bioinformatical analysis and experimental assays identified CCAAT/enhancer binding protein β (C/EBPβ) as a target gene of miR-191 in RA-FLS. Enforced expression of C/EBPβ rescued the cellular phenotypes induced by miR-191. In addition, an inverse correlation between the C/EBPβ level and hypoxia stimulation was found in RA-FLS, and overexpression of C/EBPβ could partly rescue the hypoxia-induced cell proliferation.

Conclusion: We demonstrated the miR-191-C/EBPβ signaling pathway mediating the hypoxia-induced cell proliferation in RA.

Keywords: CCAAT/enhancer binding protein β; Fibroblast-like synoviocytes; Hypoxia; Rheumatoid arthritis; miR-191.

Fig. 1

Upregulation of miR-191 in hypoxic RA-FLS. a HE staining of synovial tissue samples from RA and OA patients. b miRNA profiling analysis for RA-FLS, cultured under normal or hypoxia conditions for 24 h, identified a subset of dysregulated miRNAs. Data was represented as fold change of miRNAs expression in hypoxic RA-FLS, compared to normoxic RA-FLS. c Further validation of the miR-191, miR-297, miR-499b-3p, miR-770, and miR-936 expression in three RA-FLS cells and three OA-FLS cultured under hypoxia condition for 24 h, compared to normoxia condition by quantitative RT-PCR. Data represent results from three independent experiments, shown as means ± SEMs (n = 3, *p < 0.05 by t test)

Fig. 2

miR-191 promoted the cellular proliferation of RA-FLS. a Confirmation of the level of miR-191 in RA-FLS after transfected with mimic-miR-191 or inhibitor-miR-191 for 24 h by quantitative RT-PCR. b RA-FLS transfected with mimic-miR-191 and inhibitor miR-191 were plated at 4 × 10³ cells/well in 96-well plates; the cell viability of RA-FLS was determined by CCK8 at 0, 24, 48, and 72 h. c Increased expression of cyclinD1 in RA-FLS transfected with mimic-miR-191 for 48 h. Quantitative analysis on three repeats were presented. d Decreased expression of cyclinD1 at the protein level in RA-FLS transfected with inhibitor-miR-191 for 48 h. Quantitative analysis on three repeats was presented. e The cell cycle analysis by flow cytometry indicating the increased G₁/S transition of RA-FLS by mimic-miR-191 transfection for 48 h. f The cell cycle analysis by flow cytometry indicating the decreased G₁/S transition of RA-FLS by inhibitor-miR-191 after transfection for 48 h. All experiments were repeated three times. Data were presented as mean ± SEM (n = 3, ***p < 0.001, *p < 0.05 by t test)

Fig. 3

miR-191 inhibited apoptosis in RA-FLS. a RA-FLS were starved with 1% fetal serum for 24 h with or without overexpression of miR-191, followed by FACS analysis. b Bar graph showing the decreased apoptotic cell percentage after transfection with mimc-miR-191. c RA-FLS were starved with 1% fetal serum for 24 h with or without knockdown of miR-191, followed by FACS analysis. d Bar graph showing the increased apoptotic cell percentage after transfection with inhibitor-miR-191. e Transwell assays using RA-FLS cells after transfection with inhibitor-miR-191 for 24 h showed knockdown of miR-191 significantly suppressed the cell invasion of RA-FLS. f Quantitative analysis of e. All experiments were repeated three times. Data were presented as mean ± SEM (n = 3, *p < 0.05 by t test)

Fig. 4

miR-191 regulates C/EBPβ expression in RA-FLS. a quantitative RT-PCR analyses demonstrated the decrease of mRNA levels of C/EBPβ in RA-FLS after transfected with miR-191-mimic for 24 h. b Western blot analyses demonstrated the decrease of C/EBPβ protein level in RA-FLS after transfected with miR-191-mimic for 48 h. c Sequences of the predicted miR-191 target sequences in the 3′-UTR of C/EBPβ and its mutant containing altered nucleotides in the 3′-UTR. d Luciferase reporter assays demonstrated the inhibition of C/EBPβ 3′-UTR by direct interaction with miR-191. All experiments were repeated three times. Data were presented as mean ± SEM (n = 3, *p < 0.05 by t test)

Fig. 5

C/EBPβ mediated the miR-191 regulation of cell proliferation. a Western blot analyses demonstrated the knockdown of C/EBPβ in RA-FLS after transfection of siRNAs for 48 h. b RA-FLS transfected with NC or si-C/EBPβ2 were plated at 4 × 10³ cells/well in 96-well plates; the cell viability of RA-FLS was determined by CCK8 at 0, 36, and 72 h. c Cell cycle analyses showing the increased G₁/S transition after knockdown of C/EBPβ for 48 h. d Western blot analysis of the levels of C/EBPβ in RA-FLS after being overexpressed with C/EBPβ for 48 h. e CCK8 assay showed that overexpression of C/EBPβ rescued the cell proliferation regulation by miR-191 in RA-FLS. All experiments were repeated three times. Data were presented as mean ± SEM (n = 3, **p < 0.01, *p < 0.05 by t test)

Fig. 6

C/EBPβ involved in the regulation of hypoxia-induced cell proliferation. a quantitative RT-PCR demonstrated the decrease of the C/EBPβ mRNA level in RA-FLS cultured in 3% O₂ hypoxia for 24 h. b Western blot demonstrated the downregulation of C/EBPβ at the protein level in RA-FLS cultured in 3% O₂ hypoxia for 48 h. c Cell proliferation capacity was determined by CCK8 assay in RA-FLS overexpressed with C/EBPβ cultured under condition of normoxia or hypoxia. (A: Normoxia+pEnter-ctrl versus Hypoxia+pEnter-ctrl, p < 0.05; B: Hypoxia+pEnter-ctrl versus Hypoxia+pEnter-C/EBPβ, p < 0.05). All experiments were repeated three times. Data were presented as mean ± SEM (n = 3, *p < 0.05 by t test)