Abstract
Mesenchymal stem cell (MSC) is a type of stem cell that is capable of differentiating into osteoblasts and adipocytes. The pathological perturbation of MSC fate determination is well demonstrated by the replacement of bone tissues with fat in those with osteoporosis and osteopenia. Cell fate determination can be regulated by epigenetic and post-transcriptional mechanisms. MicroRNAs (miRNAs) are small endogenous non-coding RNA molecules that mediates the post-transcriptional regulation of genes expression. We hypothesized that miRNA specified to PPARγ, a major transcription factor of adipogenesis, is responsible for the differentiation of MSCs into osteoblasts. Candidate miRNA that is responsible for target gene inhibition was identified from the miRNA database via bioinformatic analyses. In this study, miR-130a and miR-27b were selected for investigation on their role in specifically binding to peroxisome proliferator-activated receptor γ (PPARγ) via in vitro osteogenesis of human MSCs. During osteogenic differentiation of human MSCs, the expression level of miR-130a and miR-27b were found to be upregulated. In the meanwhile, adipogenic marker genes (PPARγ and C/EBPβ) were found to decrease, which is in contrary to the increased expression of osteogenic marker genes (RUNX2 and Osterix). MSCs were transfected with mimics and inhibitors of miR-130a and miR-27b during in vitro osteogenesis followed by evaluation for the presence of osteogenic markers via quantitative gene expression, Western blot analysis and alkaline phosphatase activity assay. The overexpression of miR-130a and miR-27b is shown to enhance osteogenesis by increasing the gene expression of RUNX2 and Osterix, the protein expression of RUNX2, COL1A1, and Osterix as well as the alkaline phosphatase activity. Taken altogether, these results suggested that miR-130a and miR-27b could promote osteogenesis in human MSCs by targeting the PPARγ.
Highlights
Mesenchymal stem cells (MSCs) were first discovered by Friedenstein et al (1966)
Sequences were submitted to RegRNA, TargetScan and miRanda tools which resulted in the selection of 96, 10, and 23 predicted miRNA (Figure 1B and Supplementary Data 1)
The predicted miRNAs were again chosen according to the selection criteria and only 16 miRNA with complete match were categorized as candidate miRNAs (Supplementary Data 2)
Summary
Mesenchymal stem cells (MSCs) were first discovered by Friedenstein et al (1966). MSCs are adult multipotent stem cells that restrain multi-cell type differentiation property, in mesodermal lineages such as osteoblasts, chondrocytes, and adipocytes (Pittenger et al, 1999; Robey et al, 2015). The pivotal role of miRNAs lies in its ability to mediate the negative regulation of gene expression by base pairing with complementary sequences. This pairing downregulates the expression of target genes by inducing mRNA degradation or translational inhibition at the post-transcriptional level (Hammond et al, 2001; Bartel, 2004; He and Hannon, 2004; Valencia-Sanchez et al, 2006; Winter et al, 2009). The commitment of these two lineage is mutually unique and the understanding of this plasticity mechanism could pave the way for further understanding of osteoporosis or other bone diseases, which are caused by the infiltration of the bone tissue by adipocytes (Berendsen and Olsen, 2014)
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