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Circ_0003204 regulates the osteogenic differentiation of human adipose-derived stem cells via miR-370-3p/HDAC4 axis

ABSTRACT: Human adipose-derived stem cells (hASCs) are a promising cell type for bone tissue regeneration. Circular RNAs (circRNAs) have been shown to play a critical role in regulating various cell differentiation and involve in mesenchymal stem cell osteogenesis. However, how circRNAs regulate hASCs in osteogenesis is still unclear. Herein, we found circ_0003204 was significantly downregulated during osteogenic differentiation of hASCs. Knockdown of circ_0003204 by siRNA or overexpression by lentivirus confirmed circ_0003204 could negatively regulate the osteogenic differentiation of hASCs. We performed dual-luciferase reporting assay and rescue experiments to verify circ_0003204 regulated osteogenic differentiation via sponging miR-370-3p. We predicted and confirmed that miR-370-3p had targets in the 3′-UTR of HDAC4 mRNA. The following rescue experiments indicated that circ_0003204 regulated the osteogenic differentiation of hASCs via miR-370-3p/HDAC4 axis. Subsequent in vivo experiments showed the silencing of circ_0003204 increased the bone formation and promoted the expression of osteogenic-related proteins in a mouse bone defect model, while overexpression of circ_0003204 inhibited bone defect repair. Our findings indicated that circ_0003204 might be a promising target to promote the efficacy of hASCs in repairing bone defects.

SUBMITTER: Yu L

PROVIDER: S-EPMC9213414 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Project description:BackgroundHuman adipose-derived stem cells (hADSCs) are stem cells with the potential to differentiate in multiple directions. miR-204-5p is expressed at low levels during the osteogenic differentiation of hADSCs, and its specific regulatory mechanism remains unclear. Here, we aimed to explore the function and possible molecular mechanism of miR-204-5p in the osteogenic differentiation of hADSCs.MethodsThe expression patterns of miR-204-5p, Runx2, alkaline phosphatase (ALP), osteocalcin (OCN), forkhead box C1 (FOXC1) and growth differentiation factor 7 (GDF7) in hADSCs during osteogenesis were detected by qRT-PCR. Then, ALP and alizarin red staining (ARS) were used to detect osteoblast activities and mineral deposition. Western blotting was conducted to confirm the protein levels. The regulatory relationship among miR-204-5p, FOXC1 and GDF7 was verified by dual-luciferase activity and chromatin immunoprecipitation (ChIP) assays.ResultsmiR-204-5p expression was downregulated in hADSC osteogenesis, and overexpression of miR-204-5p suppressed osteogenic differentiation. Furthermore, the levels of FOXC1 and GDF7 were decreased in the miR-204-5p mimics group, which indicates that miR-204-5p overexpression suppresses the expression of FOXC1 and GDF7 by binding to their 3'-untranslated regions (UTRs). Overexpression of FOXC1 or GDF7 improved the inhibition of osteogenic differentiation of hADSCs induced by the miR-204-5p mimics. Moreover, FOXC1 was found to bind to the promoter of miR-204-5p and GDF7, promote the deacetylation of miR-204-5p and reduce the expression of miR-204-5p, thus promoting the expression of GDF7 during osteogenic differentiation. GDF7 induced hADSC osteogenesis differentiation by activating the AKT and P38 signalling pathways.ConclusionsOur results demonstrated that the miR-204-5p/FOXC1/GDF7 axis regulates the osteogenic differentiation of hADSCs via the AKT and p38 signalling pathways. This study further revealed the regulatory mechanism of hADSC differentiation from the perspective of miRNA regulation.

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Circ_0003204 regulates the osteogenic differentiation of human adipose-derived stem cells via miR-370-3p/HDAC4 axis

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