Long non-coding RNA HOTAIR promotes osteoarthritis progression via miR-17-5p/FUT2/β-catenin axis

Abstract

Osteoarthritis (OA) is a chronic joint disease and hard to cure at present. Accumulating evidence suggests long noncoding RNA-HOTAIR (lncRNA-HOTAIR) plays important role in OA progression. However, the underlying molecular mechanism of HOTAIR in OA progression has not been well elucidated. In the present study, we identified that HOTAIR level was upregulated in OA cartilage tissues. High expression of HOTAIR was correlated with modified Mankin scale, extracellular matrix (ECM) degradation and chondrocytes apoptosis. The expression of miR-17-5p was down-regulated, while alpha-1, 2 fucosyltransferase 2 (FUT2) was increased in OA progression. Luciferase reporter and RNA immunoprecipitation (RIP) assays indicated that HOTAIR could directly bind to miR-17-5p and indirectly upregulate FUT2 level. Functional investigation revealed HOTAIR and FUT2 aggravated ECM degradation and chondrocytes apoptosis, and this effect could be reversed by miR-17-5p. Altered FUT2 modulated the activity of wnt/β-catenin pathway and HOTAIR/miR-17-5p also mediated wnt/β-catenin pathway through FUT2. Collectively, our findings indicated that HOTAIR/miR-17-5p/FUT2 axis contributed to OA progression via wnt/β-catenin pathway, which might provide novel insights into the function of lncRNA-driven in OA.

Fig. 1. The differential expression of HOTAIR, miR-17-5p and FUT2 in clinical samples and human chondrocytes.

a The expression of HOTAIR in healthy and OA human cartilage tissues was identified by RT-qPCR and was positively correlated with Mankin scale. b The level of miR-17-5p was lower in OA cartilage tissues compared with healthy cartilage tissues by RT-qPCR and were negatively correlated with Mankin scale. c FUT2 expression was determined in OA cartilage tissues and normal cartilage tissues by RT-qPCR. The level of FUT2 was positively correlated with Mankin scale. d Comparison on the tissues by IHC, the higher level of FUT2 was observed in OA tissues. e FUT2 expression was detected by immunofluorescence in chondrocytes incubated in the presence or absence of IL-1β at 10 ng/ml for 24 h. f The inverse relationship was observed between HOTAIR and miR-17-5p in OA cartilage tissues. g The miR-17-5p level was negatively correlated with FUT2 expression in OA cartilage. The data were means ± S.D. of three independent assays (*P < 0.05)

Fig. 2. Functional investigation of HOTAIR in chondrocytes.

a Representative catabolic and anabolic proteins, MMP-13, ADAMTS-5, type II collagen and aggrecan were analyzed by western blot in human chondrocytes incubated in the presence or absence of IL-1β at 10 ng/ml for 24 h after transfection with HOTAIR or siHOTAIR. b MMP-13 and type II collagen levels in chondrocytes transfected with HOTAIR or siHOTAIR were analyzed by immunofluorescence staining. c, d Ki67 immunofluorescence staining and CCK-8 proliferation assay were used to identify the proliferative capability of chondrocytes after transfecting with HOTAIR or siHOTAIR. e, f 24 h after trasnfection and IL-1β stimulation, TUNEL assays and flow cytometry were performed to evaluate apoptosis of chondrocytes. g 24 h after trasnfection and IL-1β stimulation, Bax, cleaved caspase-3 and cleaved caspase-9 levels were measured by western blot. Each bar represented mean ± SD (n = 3). *P < 0.05 vs. control

Fig. 3. HOTAIR acted as a ceRNA by sponging miR-17-5p and regulated FUT2 expression indirectly.

a The predicted binding sites of miR-17-5p to HOTAIR sequence and the luciferase activity of chondrocytes co-transfected with miR-17-5p mimic and luciferase reporters containing HOTAIR-Wt or HOTAIR-Mut transcript were shown. b RNA immunoprecipitation was performed in chondrocytes transfected with miR-NC and miR-17-5p mimic. HOTAIR expression was detected by using qRT-PCR. RNA levels were presented as fold enrichment in Ago2 relative to IgG immunoprecipitates. c FUT2 protein was detected by western blot and immunofluorescence after transfected with HOTAIR or siHOTAIR. d The predicted binding sites of miR-17-5p to HOTAIR sequence and the luciferase activity of chondrocytes co-transfected with miR-17-5p mimic and luciferase reporters containing FUT2-Wt or FUT2-Mut transcript were shown. e FUT2 protein was detected by western blot and immunofluorescence staining after transfecting with miR-17-5p mimic or miR-17-5p inhibitor. f FUT2 protein was detected by western blot after co-transfecting with HOTAIR and miR-17-5p mimic or siHOTAIR and miR-17-5p inhibitor. The data were means ± S.D. of three independent assays (*P < 0.05)

Fig. 4. MiR-17-5p reversed HOTAIR-mediated ECM degradation.

a MMP-13, ADAMTS-5, type II collagen and aggrecan levels were detected by western blot after transfection and IL-1β stimulation. b MMP-13 and type II collagen levels were detected by immunofluorescence staining after transfection and IL-1β stimulation. Each bar represented mean ± SD (n = 3). *P < 0.05 vs. control

Fig. 5. MiR-17-5p reversed HOTAIR-mediated anti-proliferation and pro-apoptosis in IL-1β-induced chondrocytes.

a The proliferative ability of chondrocytes was measured by ki67 immunofluorescence staining. b, c Chondrocytes apoptosis was detected by TUNEL assay and Flow cytometry. Each bar represented mean ± SD (n = 3). *P < 0.05 vs. control

Fig. 6. The effects of HOTAIR/miR-17-5p/FUT2 on apoptotic molecules and cartilage degradation in vivo.

a Cleaved caspase-3, cleaved caspase-9 and Bax levels were detected by western blot after tansfection and IL-1β stimulation. b Histologic section of cartilage structure was stained by safranin-O in each group. The histologic scores were assessed using modified mankin’s scale. Each bar represented mean ± SD (n = 3). *P < 0.05 vs. control

Fig. 7. The effect of HOTAIR/miR-17-5p/FUT2 axis on wnt/β-catenin pathway.

a, b The effect of FUT2 on wnt/β-catenin pathway was detected by western blot and immunofluorescence staining. The data are means ± S.D. of three independent assays (*P < 0.05). c, d The effect of HOTAIR/miR-17-5p/FUT2 axis on wnt/β-catenin pathway was detected by western blot. The data were means ± S.D. of three independent assays (*P < 0.05)