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Mesenchymal stem cells (MSCs) are multipotent cells and their differentiation into the osteoblastic lineage is strictly controlled by several regulators, including microRNAs (miRNAs). Runx2 is a bone transcription factor required for osteoblast differentiation. Here, we used in silico analysis to identify a number of miRNAs that putatively target Runx2 and its co-factors to mediate both positive and negative regulation of osteoblast differentiation. Among these miRNAs, miR-590-5p was selected and its expression was found to be increased during osteoblast differentiation. When mouse MSCs (mMSCs) were transiently transfected with a miR-590-5p mimic, we detected an increase in both calcium deposition and the mRNA expression of osteoblast differentiation marker genes such as alkaline phosphatase (ALP) and type I collagen genes. Smad7 was found to be among the putative target genes of miR-590-5p and its mRNA and protein expression decreased after miR-590-5p mimic transfection in human osteoblast-like cells (MG63). Our analysis indicated that Runx2 was not a putative target of miR-590-5p. However, Runx2 protein, but not mRNA expression, increased after miR-590-5p mimic transfection in MG63 cells. Runx2 protein expression was increased with knockdown of Smad7 expression by Smad7 siRNA in these cells. We further identified that the 3'-untranslated region of Smad7 was directly targeted by miR-590-5p; this was done using the luciferase reporter gene system. It is known that Smad7 inhibits osteoblast differentiation via Smurf2-mediated Runx2 degradation. Hence, based on our results, we suggest that miR-590-5p promotes osteoblast differentiation by indirectly protecting and stabilizing the Runx2 protein by targeting Smad7 gene expression. J. Cell. Physiol. 232: 371-380, 2017. © 2016 Wiley Periodicals, Inc.
This article was published in the following journal.
Name: Journal of cellular physiology
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