MicroRNA-214 regulates smooth muscle cell differentiation from stem cells by targeting RNA-binding protein QKI.

Yutao Wu,Zhoubin Li,Jianhua Zhu,Bing Dai,Feng Hu,Li Zhang,Ting Chen,Feng Yang,Mei Yang

doi:10.18632/oncotarget.15189

Abstract

MicroRNA-214(miR-214) has been recently reported to regulate angiogenesis and embryonic stem cells (ESCs) differentiation. However, very little is known about its functional role in vascular smooth muscle cells (VSMCs) differentiation from ESCs. In the present study, we assessed the hypothesis that miR-214 and its target genes play an important role in VSMCs differentiation. Murine ESCs were seeded on collagen-coated flasks and cultured in differentiation medium for 2 to 8 days to allow VSMCs differentiation. miR-214 was significantly upregulated during VSMCs differentiation. miR-214 overexpression and knockdown in differentiating ESCs significantly promoted and inhibited VSMCs -specific genes expression, respectively. Importantly, miR-214 overexpression in ESCs promoted VSMCs differentiation in vivo. Quaking (QKI) was predicted as one of the major targets of miR-214, which was negatively regulated by miR-214. Luciferase assay showed miR-214 substantially inhibited wild type, but not the mutant version of QKI-3-UTR-luciferase activity in differentiating ESCs, further confirming a negative regulation role of miR-214 in QKI gene expression. Mechanistically, our data showed that miR-214 regulated VSMCs gene expression during VSMCs differentiation from ESCs through suppression of QKI. We further demonstrated that QKI down-regulated the expression of SRF, MEF2C and Myocd through transcriptional repression and direct binding to promoters of the SRF, MEF2c and Myocd genes. Taken together, we have uncovered a central role of miR-214 in ESC-VSMC differentiation, and successfully identified QKI as a functional modulating target in miR-214 mediated VSMCs differentiation.

Highlights

Regenerative medicine is an interdisciplinary field with the ultimate goal to repair, replace, or regenerate cells, tissues and organs that are lost or damaged due to disease, injury, or ageing
MiR-214 overexpression dramatically increased the expression of vascular smooth muscle cells (VSMCs) markers such as smooth muscle α-actin (SMA) and SM22 at both mRNA (Figure 1D) and protein levels (Figure 1E, 1F), whereas, miR-214 inhibition significantly downregulated the expression of VSMCs markers (Figure 1D-1F)
MiR-214 is involved in VSMCs differentiation in vivo

Summary

Introduction

Regenerative medicine is an interdisciplinary field with the ultimate goal to repair, replace, or regenerate cells, tissues and organs that are lost or damaged due to disease, injury, or ageing. Embryonic stem cells (ESCs), one of the most promising stem cell sources, are pluripotent derivatives of the inner cell mass of blastocysts They have the capacity for unlimited growth and self-renewal and the ability to differentiate into a wide range of specialized cell types including vascular endothelial cells [5, 6] and smooth muscle cells (SMCs) [7,8,9,10]. To investigate VSMCs differentiation closely, we have established an www.impactjournals.com/oncotarget excellent and simple in vitro VSMCs differentiation model from ESCs by using collagen as cellular base membrane and demonstrated that differentiation of mouse ESCs towards VSMCs lineage is mediated by a fairly complicated regulatory circuitry composing of transcription factors (Sp1 [11]) and DNA/RNA binding proteins (heterogeneous-nuclear ribonucleoprotein and A1 [12]). These findings have significantly improved our understanding regarding VSMCs differentiation and cardiovascular system development, the detailed molecular mechanisms of VSMCs differentiation from ESCs have not been fully clarified

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Results

Conclusion