The Mechanism Of Retinoic Acid In Regulating Adipogenesis And Osteogenesis Of Human Bone Marrow Mesenchymal Stem Cells

Human bone marrow mesenchymal stem cells(hBMSCs)may differentiate into osteoblasts and adipocytes.The imbalance of these two cell differentiation may be an important factor in the development of osteoporosis and other diseases.Retinoic acid(RA)is a kind of signal molecule from vitamin A in vivo,which plays an important role in embryonic development and cell differentiation.RA can activate retinoic acid receptor(RAR)and retinoic acid X receptor(RXR),which is involved in the regulation of adipogenic and osteogenic differentiation.Therefore,study of molecular mechanism of retinoic acid receptor in regulating adipogenic and osteogenic differentiation of hBMSCs,will be helpful to elucidate the pathogenesis of osteoporosis.This study was based on the high-throughput transcriptome sequencing and bioinformatics analysis,combined with the specific function experiments,to illustrate different effects of the two types of retinoic acid receptors(RAR and RXR)during adipogenesis and osteogenesis in hBMSCs.Part ?:The transcriptome analysis of adipogenesis of human bone marrow mesenchymal stem cellsObjective:To clarify the possible role of retinoic acid signaling pathway in adipogenesis in hBMSCs by RNA sequencing and bioinformatics analysis.Methods:Human bone marrow mesenchymal stem cells were cultured and identified in vitro,and then induced into adipogenic differentiation.RNA sequencing was carry out at multiple time points during adipogenesis,the sequencing results were analysized by Weighted Gene Co-Expression Network Analysis(WGCNA)method.Results:WGCNA based on transcriptome sequencing data during adipogenesis showed that RAR,PI3K-AKT,Hippo,TGFbeta,PPAR signaling pathways and so on were located in Turquoise module,the module is involved in the initiation of adipogenic differentiation,and is most related with hBMSCs sample.RAR is a kind of negative regulatory signal in this module.RXR related gene RXRA is located in the Blue module which is most related to the sample which is induced to adipogenic differentiation for seven days,and is involved in the development process of adipogenic differentiation.RXRA is a positive regulatory signal in this module.The signal pathways correlated significantly with RAR in Turquoise module included Focal adhesion,Regulation of actin cytoskeleton,ECM-receptor interaction,Cytokine-cytokine receptor interaction,TNF,PI3K-Akt,Jak-STAT,VEGF,Hippo,Rap1,MAPK and Ras.RAR may inhibit adipogenic differentiation through these pathways.The signal pathways correlated significantly with RXR in Blue module included HIF-1,PI3K-AKT and estrogen signaling pathway,as well as a variety of lipid and amino acid metabolism pathway,suggesting that RXRA may promote the conversion of cell phenotype through metabolic regulation.Conclusion:RAR may be a negative regulatory signal for initiation of adipogenic differentiation,and RXR may be a positive regulatory signal for adipogenic differentiation,which promotes further development of adipogenic differentiation.Part ?:The function study of retinoic acid on the adipogenic and osteogenic differentiation of human bone marrow mesenchymal stem cellsObjective:To illuminate the molecular mechanism of RAR and RXR in regulating the osteogenic and adipogenic differentiation of hBMSCs by functional studies.Methods:hBMSCs were treated with retinoic acid receptors(RAR and RXR)agonists or antagonists during adipogenic and osteogenic differentiation,the cell phenotypes were determined using oil red O staining and alizarin red staining.The expression levels of adipogenic genes,TGFbeta/SMAD and Wnt signal pathway related gene were determined by qRT-PCR and Western blotting.The promoter activity of transcription factors were examined using a luciferase reporter assay.Knockdown of the target genes were executed by using shRNA lentiviral particles.Results:RAR promoted adipogenic differentiation in the early phase but inhibited differentiation at a later stage,involving TGFbeta/SMAD signaling pathway and Wnt signaling pathway,RAR inhibited promoter activity of adipogenic transcription factor C/EBPbeta and PPARgamma.The major subtype mediating RAR effects is RARbeta.RXR promoted adipogenesis in hBMSCs,but inhibited it in SW872 cells,in a cell type-dependend manner.RAR agonists significantly increased the expression of BMP2,OSX,RUNX2,OCN and OPN,but decreased the expression of ALPL,CollAl and ON,and inhibited calcium nodule formation.RXR agonist inhibited slightly the expression of ALPL,CollAl and ON,increased slightly the expression of OPN,and no obvious effects on the expression of BMP2,OSX,RUNX2 and OCN.RXR promoted adipogenic differentiation of hBMSCs during osteogenic induction.Conclusion:RAR and RXR show different effects and molecular mechanisms on adipogenic and osteogenic differentiation of hBMSCs.Part ?:Transcriptome-wide analysis of retinoic acid on the adipogenic and osteogenic differentiation in human bone marrow mesenchymal stem cellsObjective:To investigate the role of RAR and RXR in the regulation of adipogenic and osteogenic differentiation of hBMSCs at transcriptome scale.Methods:The mRNA,lncRNA and miRNA expression profiles on adipogenesis and osteogenesis in hBMSCs with RAR agonists and RXR agonists treatment were obtained using RNA-seq.The transcriptome data were analyzed using bioinformatics methods.miRNAs and their target genes regulated by RAR or RXR were screened.The miRNA-mRNA interaction network were constructed.Results:RNA-seq data showed RAR agonist upregulated Focal adhesion?Hippo?Rapl?PI3K-AKT?Wnt?TGF? signaling pathway and so on,downregulated PPAR signal pathway.RXR upregulated PPAR signal pathway,and was closely related to numerous lipid and amino acid metabolic pathways.The lncRNAs,miRNAs and corresponding target genes regulated by RAR or RXR were significantly different.Conclusion:RAR is a signal regulating cell differentiation,which regulates the adipogenic and osteogenic differentiation in hBMSCs,involving a variety of signaling pathways,IncRNAs and miRNAs.RXR is a signal regulating cell metabolism,and is closely related to intracellular lipid metabolism and amino acid metabolism.