MiR-378a-5p Regulates Proliferation and Migration in Vascular Smooth Muscle Cell by Targeting CDK1.

Shaoyan Liu,Hui Xin,Rui Zhang,Amara Lobo,Yanyan Yang,Ningning Tang,Shaoyan Jiang,Tao Yu,Song Liu,Hong Xu

doi:10.3389/fgene.2019.00022

Abstract

Objective: Abnormal proliferation or migration of vascular smooth muscle cells (VSMCs) can lead to vessel lesions, resulting in atherosclerosis and in stent-restenosis (IRS). The purpose of our study was to establish the role of miR-378a-5p and its targets in regulating VSMCs function and IRS.Methods: EdU assays and Cell Counting Kit-8 (CCK-8) assays were applied to evaluate VSMCs proliferation, wound healing assays and transwell assays were applied to assess cells migration. Furthermore, quantitative reverse transcription–polymerase chain reaction (qRT-PCR) was performed to investigate the expression level of miR-378a-5p IRS patients and healthy individuals. Target genes were predicted using Target Scan and miRanda software, and biological functions of candidate genes were explored through bioinformatics analysis. Moreover, RNA-binding protein immunoprecipitation (RIP) was carried out to analyze the miRNAs interactions with proteins. We also used Immunofluorescence (IF) and fluorescence microscopy to determine the binding properties, localization and expression of miR-378a-5p with downstream target CDK1.Results: The expression of miR-378a-5p was increased in the group with stent restenosis compared with healthy people, as well as in the group which VSMCs stimulated by platelet-derived growth factor-BB (PDGF-BB) compared with NCs. MiR-378a-5p over-expression had significantly promoted proliferative and migratory effects, while miR-378a-5p inhibitor suppressed VSMC proliferation and migration. CDK1 was proved to be the functional target of miR-378a-5p in VSMCs. Encouragingly, the expression of miR-378a-5p was increased in patients with stent restenosis compared with healthy people, as well as in PDGF-BB-stimulated VSMCs compared with control cells. Furthermore, co-transfection experiments demonstrated that miR-378a-5p over-expression promoted proliferation and migration of VSMCs specifically by reducing CDK1 gene expression levels.Conclusion: In this investigatory, we concluded that miR-378a-5p is a critical mediator in regulating VSMC proliferation and migration by targeting CDK1/p21 signaling pathway. Thereby, interventions aimed at miR-378a-5p may be of therapeutic application in the prevention and treatment of stent restenosis.

Highlights

Coronary artery disease (CAD) is a serious disease threatening human health with its high mortality rate
MiR-378a-5p expression were detected between the patients with stent-restenosis and control group, respectively, by qRT-PCR, in which we found that miR-378a-5p expression levels were upregulated in stent-restenosis patients compared with control group (Figure 1A); MiR-378a-5p expression levels were higher in atherosclerotic plaques of ApoE knockout (ApoE -/-) mice than in control subjects as measured by qRT-PCR (Figure 1B)
We identified that miR-378a-5p is an important modulator in the PDGF-BB stimulated proliferation and migration of Vascular smooth muscle cells (VSMCs) by targeting, at least partly Cyclin-dependent kinase 1 (CDK1) pathway

Summary

Introduction

Coronary artery disease (CAD) is a serious disease threatening human health with its high mortality rate. There will be new atherosclerosis around stent implantation, and this will further increase the rate of restenosis after stent implantation (Wang H. et al, 2015; Liu et al, 2018). Endothelialization occurs gradually which cannot be removed from the blood vessels again; so, if in-stent restenosis occurs, stent implantation must be repeated (Alfonso et al, 2006; Finn et al, 2007). When VSMCs are stimulated, they promote the transfer of VSMCs from tunica media to tunica intima, from the contractile to the secretory, while stimulating free VSMCs and fibroblasts to secrete a large amount of extracellular matrix; the extracellular matrix is continuously deposited in the blood vessels, causing the intima to gradually thicken, resulting in stenosis (BraunDullaeus et al, 1998). Investigating key regulators and understanding the molecular mechanisms of VSMC biology has become a major method of treating atherosclerosis and stent restenosis

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Discussion

Conclusion