MicroRNA‐590‐5p Stabilizes Runx2 by Targeting Smad7 During Osteoblast Differentiation
MicroRNA‐590‐5p Stabilizes Runx2 by Targeting Smad7 During Osteoblast Differentiation
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DOI：
10.1002/jcp.25434
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发表时间：
2017-02
影响因子：
5.6
通讯作者：
M. Vishal;S. Vimalraj;R. Ajeetha;M. Gokulnath;R. Keerthana;Z. He;N. Partridge;N. Selvamurugan
中科院分区：
文献类型：
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作者：
M. Vishal;S. Vimalraj;R. Ajeetha;M. Gokulnath;R. Keerthana;Z. He;N. Partridge;N. Selvamurugan
间充质干细胞（MSC）是多能细胞，并且它们向成骨细胞谱系的分化受到包括microRNA（miRNAs）在内的若干调节因子的严格控制。Runx 2是成骨细胞分化所需的骨转录因子。在这里，我们使用计算机分析来鉴定许多miRNA，这些miRNA靶向Runx 2及其辅因子，以介导成骨细胞分化的正向和负向调节。在这些miRNA中，选择了miR-590 - 5 p，发现其表达在成骨细胞分化期间增加。当用miR-590 - 5 p模拟物瞬时转染小鼠MSC（mMSC）时，我们检测到钙沉积和成骨细胞分化标记基因（如碱性磷酸酶（ALP）和I型胶原基因）的mRNA表达增加。Smad 7被发现是miR-590 - 5 p的推定靶基因之一，并且在人成骨样细胞（MG 63）中miR-590 - 5 p模拟物转染后其mRNA和蛋白表达降低。我们的分析表明Runx 2不是miR-590 - 5 p的假定靶点。然而，在MG 63细胞中，miR-590 - 5 p模拟物转染后，Runx 2蛋白而不是mRNA表达增加。在这些细胞中，Runx 2蛋白表达随着Smad 7 siRNA敲低Smad 7表达而增加。我们进一步确定Smad 7的3′-非翻译区直接被miR-590 - 5 p靶向;这是使用荧光素酶报告基因系统完成的。已知Smad 7通过Smurf 2介导的Runx 2降解抑制成骨细胞分化。因此，基于我们的研究结果，我们认为miR-590 - 5 p通过靶向Smad 7基因表达间接保护和稳定Runx 2蛋白来促进成骨细胞分化。J.细胞。232：371-380，2017。© 2016 Wiley Periodicals，Inc.
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.