miR-193b-5p regulates chondrocytes metabolism by directly targeting histone deacetylase 7 in interleukin-1ß-induced osteoarthritis.

There is increasing evidence regarding the pivotal roles of microRNAs (miRNAs) and histone deacetylases (HDACs) in the development of osteoarthritis (OA). This study aimed to determine whether miR-193b-5p regulates HDAC7 expression directly to affect cartilage degeneration. Expression levels of miR-193b-5p, HDAC7, matrix metalloproteinase 3 (MMP3), and MMP13 were determined in normal and OA cartilage and primary human chondrocytes (PHCs) stimulated with interleukin-1ß (IL-1ß). PHCs were transfected with a miR-193b-5p mimic or inhibitor to verify whether miR-193b-5p influences the expression of HDAC7 and MMPs. A luciferase reporter assay was performed to demonstrate the binding between miR-193b-5p and the 3'-untranslated region (UTR) of HDAC7. Expression of miR-193b-5p was reduced in IL-1ß-stimulated PHCs and in OA cartilage compared to that in normal cartilage. Luciferase reporter assay exhibited the repressed activity of the reporter construct containing the 3'UTR of HDAC7. Both miR-193b-5p overexpression and HDAC7 inhibition decreased the expression of MMP3 and MMP13, whereas the inhibition of miR-193b-5p enhanced HDAC7, MMP3, and MMP13 expression. miR-193b-5p downregulates HDAC7 directly and, as a result, inhibits MMP3 and MMP13 expression, which suggests that miR-193b-5p has a protective role in OA.

CDK6 and miR-320c Co-Regulate Chondrocyte Catabolism Through NF-κB Signaling Pathways.

BACKGROUND/AIMS: Cyclin-dependent kinase 6 (CDK6) regulates inflammatory response and cell differentiation. This study sought to determine whether CDK6 and miR-320c co-regulate chondrogenesis and inflammation. METHODS: Utilizing quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC), CDK6 and miR-320c expression were assessed in a micromass culture of human bone mesenchymal stem cells that underwent chondrogenesis in vitro as well as in chondrocytes from E16.5 mouse forelimbs. Normal chondrocytes were transfected with miR-320c mimic, miR-320c inhibitor, or CDK6-siRNA. Luciferase reporter assay results confirmed that miR-320c directly targets CDK6 by interacting with the 3'-untranslated region (3'-UTR) of its mRNA. qRT-PCR, Western blotting, and Cell Counting Kit-8 were subsequently used to evaluate the effects of miR-320c overexpression and CDK6 inhibition on inflammatory factor expression, as well as to investigate the effects of NF-kB and MAPK signaling pathway activation on IL-1ß-induced chondrocyte inflammation. RESULTS: Our results show that miR-320c expression increased during the middle stage and decreased during the late stage of hBMSC chondrogenic differentiation. In contrast, CDK6 expression decreased during the middle stage and increased during the late stage of hBMSC chondrogenic differentiation. Moreover, CDK6 expression increased in severe OA cartilage and in hypertrophic chondrocytes of mouse forelimbs at E16.5. Results of the luciferase reporter assay showed that miR-320c modulated CDK6 expression by binding to the 3'-UTR of its mRNA. miR-320c overexpression and CDK6 inhibition repressed IL-1ß-induced expression of inflammatory factors and regulated the NF-kB signaling pathway. CONCLUSION: CDK6 and miR-320c co-regulate hBMSC chondrogenesis and IL-1ß-induced chondrocyte inflammation through the NF-kB signaling pathway, suggesting that miR-320c and CDK6 inhibitors can be used to repress catabolism in human chondrocytes.

Exosomes derived from miR-92a-3p-overexpressing human mesenchymal stem cells enhance chondrogenesis and suppress cartilage degradation via targeting WNT5A.

BACKGROUND: WNT5A is known to be involved in the pathogenesis of osteoarthritis. This study investigated the molecular mechanism of exosomal miR-92a-3p and WNT5A in chondrogenesis and cartilage degeneration. METHODS: Exosomal miR-92a-3p expression was assessed in vitro in a human mesenchymal stem cell (MSC) model of chondrogenesis and in normal and OA primary human chondrocytes (PHCs). MSCs and PHCs were treated with exosomes derived from MSC-miR-92a-3p (MSC-miR-92a-3p-Exos) or its antisense inhibitor (MSC-anti-miR-92a-3p-Exos), respectively. Small interfering RNAs (siRNAs) and luciferase reporter assay were used to reveal the molecular role of exosomal miR-92a-3p and WNT5A in chondrogenesis. The protective effect of exosomes in vivo was measured using Safranin-O and Fast Green staining and immunohistochemical staining. RESULTS: Exosomal miR-92a-3p expression was elevated in the MSC chondrogenic exosome, while it was significantly reduced in the OA chondrocyte-secreted exosome compared with normal cartilage. Treatment with MSC-miR-92a-3p-Exos promoted cartilage proliferation and matrix genes expression in MSCs and PHCs, respectively. In contrast, treatment with MSC-anti-miR-92a-3p-Exos repressed chondrogenic differentiation and reduced cartilage matrix synthesis by enhancing the expression of WNT5A. Luciferase reporter assay demonstrated that miR-92a-3p suppressed the activity of a reporter construct containing the 3'-UTR and inhibited WNT5A expression in both MSCs and PHCs. MSC-miR-92a-3p-Exos inhibit cartilage degradation in the OA mice model. CONCLUSIONS: Our results suggest that exosomal miR-92a-3p regulates cartilage development and homeostasis by directly targeting WNT5A. This indicates that exosomal miR-92a-3p may act as a Wnt inhibitor and exhibits potential as a disease-modifying osteoarthritis drug.

MicroRNA-193b-3p regulates chondrogenesis and chondrocyte metabolism by targeting HDAC3.

Histone deacetylase 3 (HDAC3) plays a pivotal role in the repression of cartilage-specific gene expression in human chondrocytes. The aim of this study was to determine whether microRNA-193b-3p (miR-193b-3p) regulates the expression of HDAC3 during chondrogenesis and chondrocyte metabolism. Methods: miR-193b-3p expression was assessed in a human mesenchymal stem cell (hMSC) model of chondrogenesis, in interleukin-1ß (IL-1ß)-treated primary human chondrocytes (PHCs), and in non-degraded and degraded cartilage. hMSCs and PHCs were transfected with miR-193b-3p or its antisense inhibitor. A direct interaction between miR-193b-3p and its putative binding site in the 3'-untranslated region (3'-UTR) of HDAC3 mRNA was confirmed by performing luciferase reporter assays. Chondrocytes were transfected with miR-193b-3p before performing a chromatin immunoprecipitation assay with an anti-acetylated histone H3 antibody. To investigate miR-193b-3p-transfected PHCs in vivo, they were seeded in tricalcium phosphate-collagen-hyaluronate (TCP-COL-HA) scaffolds, which were then implanted in nude mice. In addition, plasma exosomal miR-193b-3p in samples from normal controls and patients with osteoarthritis (OA) were measured. Results: miR-193b-3p expression was elevated in chondrogenic and hypertrophic hMSCs, while expression was significantly reduced in degraded cartilage compared to non-degraded cartilage. In addition, miR-193b-3p suppressed the activity of reporter constructs containing the 3'-UTR of HDAC3, inhibited HDAC3 expression, and promoted histone H3 acetylation in the COL2A1, AGGRECAN, COMP, and SOX9 promoters. Treatment with the HDAC inhibitor trichostatin A (TSA) increased cartilage-specific gene expression and enhanced hMSCs chondrogenesis. TSA also increased AGGRECAN expression and decreased MMP13 expression in IL-1ß-treated PHCs. Further, 8 weeks after implanting PHC-seeded TCP-COL-HA scaffolds subcutaneously in nude mice, we found that miR-193b overexpression strongly enhanced in vivo cartilage formation compared to that found under control conditions. We also found that patients with OA had lower plasma exosomal miR-193b levels than control subjects. Conclusions: These findings indicate that miR-193b-3p directly targets HDAC3, promotes H3 acetylation, and regulates hMSC chondrogenesis and metabolism in PHCs.

MicroRNA-193b-3p regulates matrix metalloproteinase 19 expression in interleukin-1ß-induced human chondrocytes.

Micro(mi)RNAs are small, non-coding RNA molecules known to play a significant role in osteoarthritis (OA) initiation and development, and similar to matrix metalloproteinases (MMPs), they participate in cartilage degeneration and cleave multiple extracellular matrices. The aim of this study was to determine whether the expression of MMP-19 in interleukin (IL)-1ß-induced human chondrocytes is directly regulated by miR-193b-3p. Expression levels of miR-193b-3p and MMP-19 in normal and osteoarthritis (OA) human cartilage, and interleukin-1 ß (IL-1ß)-induced human chondrocytes were determined by real-time polymerase chain reaction. Additionally, expression level of MMP-19 in IL-1ß-induced human chondrocytes was estimated by Western blotting and immunohistochemistry analyses. The effect of miR-193b-3p on MMP-19 expression was evaluated using transient transfection of normal human chondrocytes with miR-193b-3p mimic or its antisense inhibitor (miR-193b-3p inhibitor), and siMMP-19. The putative binding site of miR-193b-3p in the 3'-untranslated region (UTR) of MMP-19 mRNA was validated by luciferase reporter assay. miR-193b-3p expression was reduced in OA cartilage compared to that in normal chondrocytes, while the opposite was observed for MMP-19. Upregulation of MMP-19 expression was correlated with downregulation of miR-193b-3p in IL-1ß-stimulated normal chondrocytes. Increase in miR-193b-3p levels was associated with silencing of MMP-19. Overexpression of miR-193b-3p suppressed the activity of the reporter construct containing the 3'-UTR of human MMP-19 mRNA and inhibited the IL-1ß-induced expression of MMP-19 and iNOS in chondrocytes, while treatment with miR-193b-3p inhibitor enhanced MMP-19 expression. MiR-193b-3p is an important regulator of MMP-19 in human chondrocytes and may relieve the inflammatory response in OA.