The miR-204-5p/FOXC1/GDF7 axis regulates the osteogenic differentiation of human adipose-derived stem cells via the AKT and p38 signalling pathways

You Zhou; Siyu Liu; Wei Wang; Qiang Sun; Mengzhu Lv; Shude Yang; Shuang Tong; Shu Guo

doi:10.1186/s13287-020-02117-4

The miR-204-5p/FOXC1/GDF7 axis regulates the osteogenic differentiation of human adipose-derived stem cells via the AKT and p38 signalling pathways

Stem Cell Res Ther. 2021 Jan 18;12(1):64. doi: 10.1186/s13287-020-02117-4.

Authors

You Zhou¹, Siyu Liu¹, Wei Wang², Qiang Sun¹, Mengzhu Lv¹, Shude Yang¹, Shuang Tong¹, Shu Guo³

Affiliations

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.

Keywords: Forkhead box C1; Growth differentiation factor 7; Human adipose-derived stem cells; Osteogenic differentiation; miR-204-5p.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bone Morphogenetic Proteins
Cell Differentiation
Cells, Cultured
Forkhead Transcription Factors / genetics
Humans
MicroRNAs* / genetics
Osteogenesis* / genetics
Proto-Oncogene Proteins c-akt / genetics
Stem Cells

Substances

Bone Morphogenetic Proteins
FOXC1 protein, human
Forkhead Transcription Factors
MIRN204 microRNA, human
MicroRNAs
growth differentiation factor 7
Proto-Oncogene Proteins c-akt