MicroRNAs comprise a group of non-coding small RNAs (17-25 nt) involved in post-transcriptional regulation that have been identified in various plants and animals. Studies have demonstrated that miRNAs are associated with stem cell self-renewal and differentiation and play a key role in controlling stem cell activities. However, the identification of specific miRNAs and their regulatory roles in the differentiation of multipotent mesenchymal stromal cells (MSCs) have so far been poorly defined. We isolated and cultured human MSCs and osteo-differentiated MSCs from four individual donors. miRNA expression in MSCs and osteo-differentiated MSCs was investigated using miRNA microarrays. miRNAs that were commonly expressed in all three MSC preparations and miRNAs that were differentially expressed between MSCs and osteo-differentiated MSCs were identified. Four underexpressed (hsa-miR-31, hsa-miR-106a, hsa-miR-148a, and hsa-miR-424) and three novel overexpressed miRNAs (hsa-miR-30c, hsa-miR-15b, and hsa-miR-130b) in osteo-differentiated MSCs were selected and their expression were verified in samples from the fourth individual donors. The putative targets of the miRNAs were predicted using bioinformatic analysis. The four miRNAs that were underexpressed in osteo-differentiated MSCs were predicted to target RUNX2, CBFB, and BMPs, which are involved in bone formation; while putative targets for miRNAs overexpressed in osteo-differentiated MSCs were MSC maker (e.g., CD44, ITGB1, and FLT1), stemness-maintaining factor (e.g., FGF2 and CXCL12), and genes related to cell differentiation (e.g., BMPER, CAMTA1, and GDF6). Finally, hsa-miR-31 was selected for target verification and function analysis. The results of this study provide an experimental basis for further research on miRNA functions during osteogenic differentiation of human MSCs.

Our results suggest that miR-92a-3p regulates cartilage development and homeostasis, which directly targets HDAC2, indicating histone hyperacetylation plays an important role in increased expression of cartilage matrix.

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-1β (IL-1β)-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-1β-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.

MicroRNAs play critical roles in the pathogenesis of osteoarthritis, the most common chronic degenerative joint disease. Exosomes derived from miR‐95‐5p‐overexpressing primary chondrocytes (AC‐miR‐95‐5p) may be effective in treating osteoarthritis. Increased expression of HDAC2/8 occurs in the tissues and chondrocyte‐secreted exosomes of patients with osteoarthritis and mediates cartilage‐specific gene expression in chondrocytes. We have been suggested that exosomes derived from AC‐miR‐95‐5p (AC‐miR‐95‐5p‐Exos) would enhance chondrogenesis and prevent the development of osteoarthritis by directly targeting HDAC2/8. Our in vitro experiments showed that miR‐95‐5p expression was significantly lower in osteoarthritic chondrocyte‐secreted exosomes than in normal cartilage. Treatment with AC‐miR‐95‐5p‐Exos promoted cartilage development and cartilage matrix expression in mesenchymal stem cells induced to undergo chondrogenesis and chondrocytes, respectively. In contrast, co‐culture with exosomes derived from chondrocytes transfected with an antisense inhibitor of miR‐95‐5p (AC‐anti‐miR‐95‐5p‐Exos) prevented chondrogenic differentiation and reduced cartilage matrix synthesis by enhancing the expression of HDAC2/8. MiR‐95‐5p suppressed the activity of reporter constructs containing the 3ʹ‐untranslated region of HDAC2/8, inhibited HDAC2/8 expression and promoted cartilage matrix expression. Our results suggest that AC‐miR‐95‐5p‐Exos regulate cartilage development and homoeostasis by directly targeting HDAC2/8. Thus, AC‐miR‐95‐5p‐Exos may act as an HDAC2/8 inhibitor and exhibit potential as a disease‐modifying osteoarthritis drug.

Osteoarthritis, also known as degenerative arthritis or degenerative joint disease, causes pain and disability worldwide. Cartilage regeneration is key to finding a cure for this disease. Adipose-derived stem cells (ASCs) are capable of differentiating into cartilage lineages in vitro and they have shown promise in the field of regenerative medicine. However, the underlying mechanisms remain unclear. In this study, we demonstrated that miR-194 levels gradually decreased during the chondrogenic differentiation of human ASCs (hASCs). After predicting the target of miR-194 using Pictar and Targetscan, we hypothesized that Sox5 is potentially the key link between miR-194 and the chondrogenesis of ASCs. Initially, we demonstrated that Sox5 is a target of miR194 according to luciferase assay analysis. We further demonstrated that the differentiation of ASCs can be controlled by miR-194 through gain or loss of function experiments, and we observed that the down-regulation of miR-194 increases its direct target gene, Sox5, and results in enhanced chondrogenic differentiation of hASCs, whereas up-regulation decreases Sox5 and inhibits chondrogenesis. We also found that miR-194 correlates with Sox5 in osteoarthritis. These findings, taken together, are the first to illustrate the critical role of miR-194 in hASC chondrogenesis, and may provide novel insight beneficial to cell manipulation methods during cartilage regeneration.