You Zhou1, Siyu Liu1, Wei Wang2, Qiang Sun1, Mengzhu Lv1, Shude Yang1, Shuang Tong1, Shu Guo3. 1. Department of Plastic Surgery, The First Hospital of China Medical University, NO 155 Nanjing street Heping Strict, Shenyang, 110001, Liaoning Province, China. 2. Institute of Respiratory Disease, The First Hospital of China Medical University, NO 155 Nanjing street Heping Strict, Shenyang, 110001, Liaoning Province, China. 3. Department of Plastic Surgery, The First Hospital of China Medical University, NO 155 Nanjing street Heping Strict, Shenyang, 110001, Liaoning Province, China. sguo@cmu.edu.cn.
Abstract
BACKGROUND: Human 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. METHODS: The 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. RESULTS: miR-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. CONCLUSIONS: Our 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.
BACKGROUND:Human 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. METHODS: The 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. RESULTS:miR-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. CONCLUSIONS: Our 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.
Authors: N M Wolfman; G Hattersley; K Cox; A J Celeste; R Nelson; N Yamaji; J L Dube; E DiBlasio-Smith; J Nove; J J Song; J M Wozney; V Rosen Journal: J Clin Invest Date: 1997-07-15 Impact factor: 14.808
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