CircPRKCH modulates extracellular matrix formation and metabolism by regulating the miR-145/HGF axis in osteoarthritis

Abstract

Background: Osteoarthritis (OA) is a chronic degenerative joint disease. Extracellular matrix (ECM) degradation is essential for OA progression. Previous studies have shown that circular RNAs (circRNAs) are involved in the pathological process of OA. CircPRKCH has been shown to be upregulated in OA chondrocytes. The present study was aimed to explore the roles of circPRKCH in vivo and in vitro models of OA and its underlying molecular mechanisms.

Methods: IL-1β-induced chondrocytes and mice injected with monosodium iodoacetate were used as OA models in vitro and in vivo, respectively. RT-qPCR was performed to measure the expression of circPRKCH, miR-145, and HGF in cartilage tissues and chondrocytes. The interaction between miR-145 and circPRKCH or HGF was verified by a dual-luciferase reporter assay. Chondrocyte apoptosis, viability, and ECM-related proteins were examined by flow cytometry, MTT assay, and Western blotting, respectively. Histopathological changes were detected by HE and Safranin O-fast green staining.

Results: The expression of circPRKCH and HGF was increased in OA cartilage tissues and IL-1β-treated chondrocytes, while miR-145 expression was decreased. IL-1β induced chondrocyte apoptosis and ECM degradation in chondrocytes. Moreover, circPRKCH promoted HGF expression and activated HGF/c-MET by directly binding to miR-145. miR-145 knockdown or HGF overexpression significantly reversed circPRKCH knockdown-mediated inhibition of apoptosis and ECM degradation in IL-1β-induced chondrocytes. Besides, miR-145 overexpression alleviated IL-1β-induced chondrocyte apoptosis and ECM degradation by inhibiting HGF/c-MET. Finally, circPRKCH knockdown reduced ECM degradation by regulating the miR-145/HGF axis in an experimental OA model in mice.

Conclusion: Our study demonstrated that circPRKCH promoted chondrocyte apoptosis and ECM degradation via the miR-145/HGF axis in OA, which may provide a novel target for OA treatment.

Keywords: CircPRKCH; Extracellular matrix; Hepatocyte growth factor; Osteoarthritis; miR-145.

Fig. 1

CircPRKCH and HGF were increased, but miR-145 was decreased in OA cartilage tissues. A–C RT-qPCR was used to detect circPRKCH, miR-145, and HGF expression in cartilage tissues of OA patients (n=16) and normal patients (n=10). D, E Western blotting was used to test HGF levels in OA patients and normal patients. Data are from three independent experiments. * p< 0.05 and ** p< 0.01

Fig. 2

CircPRKCH and HGF were increased, but miR-145 was decreased in IL-1β-treated chondrocytes. Primary human chondrocytes were isolated and treated with 10 ng/ml IL-1β to induce an OA cell model. A MTT assay was used to examine chondrocyte viability in different time points. B, C Flow cytometry was used to analyze chondrocytes apoptosis. D, E Western blotting was used to detect MMP-3, MMP-13, Aggrecan, and Collagen II expression. F RT-qPCR was used to detect the expression levels of circPRKCH, miR-145, and HGF levels. G, H Western blotting was used to test the protein levels of HGF, p-c-MET, and c-MET. Data are the means ± SD for three independent experiments. *p< 0.05, **p< 0.01, and ***p< 0.001

Fig. 3

CircPRKCH activated the HGF/c-MET signaling pathway by targeting miR-145 in chondrocytes. A, B The binding sites between circPRKCH and miR-145, and miR-145 and HGF predicted by bioinformatics tools StarBase and Targetscan, respectively. C, D The luciferase activity of wild-type (WT-circPRKCH, WT-HGF) and mutant (MUT-circPRKCH, MUT-HGF) co-transfected with miR-145 mimics or mimics NC in chondrocytes. E Chondrocytes were transfected with sh-circPRKCH or sh-NC. RT-qPCR measured circPRKCH, miR-145, and HGF levels. F Chondrocytes were transfected with miR-145 mimics, miR-145 inhibitor, or negative controls. MiR-145 and HGF expression was detected by RT-qPCR. G, H Chondrocytes were treated with sh-circPRKCH, miR-145 mimics, or miR-145 inhibitor. Western blotting was used to test HGF, p-c-MET, and c-MET expression. Data are the means ± SD for three independent experiments. *p< 0.05, **p< 0.01, and ***p< 0.001

Fig. 4

MiR-145 inhibitor or HGF overexpression reversed the effect of circPRKCH knockdown on cell apoptosis and ECM degradation. Chondrocytes were transfected with sh-circPRKCH, miR-145 inhibitor, HGF overexpressing vector, and negative controls and treated with IL-1β. A, B RT-qPCR and Western blotting were used to detect HGF level in chondrocytes. C, D Flow cytometry was used to analyze chondrocyte apoptosis. E, F Western blotting was used to determine MMP-3, MMP-13, Aggrecan, and Collagen II levels. Data are the means ± SD for three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 5

MiR-145 overexpression inhibited ECM degradation by inhibiting the HGF/c-MET signaling pathway. Chondrocytes were transfected with miR-145 mimics or HGF overexpressing vector and treated with IL-1β. A, B Flow cytometry was used to determine chondrocyte apoptosis. C, D Western blot assay was used to measure MMP-3, MMP-13, Aggrecan, and Collagen II levels. Data are the means ± SD for three independent experiments. **p< 0.01 and ***p< 0.001

Fig. 6

Knockdown of circPRKCH alleviated ECM degradation in an OA mouse model by regulating the miR-145/HGF axis. An OA mouse model was established by injecting sodium iodoacetate into the joint cavity of mice. Four groups were divided: Control, OA, OA+sh-NC, and OA+sh-circPRKCH. A HE and Safranin O-Fast Green staining were used to evaluate the osteoarthritis pathological changes and cartilage destruction. B–D RT-qPCR was used to detect circPRKCH, miR-145, and HGF levels. E, F Western blotting was used to measure HGF, p-c-MET, c-MET, MMP-3, MMP-13, Aggrecan, and Collagen II levels in mouse cartilage tissues. Data are the means ± SD for three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 7

The schematic diagram of circPRKCH regulation mechanism in OA. CircPRKCH promoted c-MET phosphorylation and activated the HGF/c-MET signaling pathway by directly binding miR-145 to disinhibit HGF, leading to ECM degradation and OA occurrence