High-content analysis of microRNAs involved in the phenotype regulation of vascular smooth muscle cells

Jian Zhang,Holger Erfle,Michael Keese,Nuh Rahbari,Kejia Kan,Zhaohui Wang,Vytaute Starkuviene,Manuel Gunkel,Carsten Sticht,Ziwei Zeng

doi:10.1038/s41598-022-07280-7

Abstract

In response to vascular injury vascular smooth muscle cells (VSMCs) alternate between a differentiated (contractile) and a dedifferentiated (synthetic) state or phenotype. Although parts of the signaling cascade regulating the phenotypic switch have been described, the role of miRNAs is still incompletely understood. To systematically address this issue, we have established a microscopy-based quantitative assay and identified 23 miRNAs that induced contractile phenotypes when over-expressed. These were then correlated to miRNAs identified from RNA-sequencing when comparing cells in the contractile and synthetic states. Using both approaches, six miRNAs (miR-132-3p, miR-138-5p, miR-141-3p, miR-145-5p, miR-150-5p, and miR-22-3p) were filtered as candidates that induce the phenotypic switch from synthetic to contractile. To identify potentially common regulatory mechanisms of these six miRNAs, their predicted targets were compared with five miRNAs sharing ZBTB20, ZNF704, and EIF4EBP2 as common potential targets and four miRNAs sharing 16 common potential targets. The interaction network consisting of these 19 targets and additional 18 hub targets were created to facilitate validation of miRNA-mRNA interactions by suggesting the most plausible pairs. Furthermore, the information on drug candidates was integrated into the network to predict novel combinatorial therapies that encompass the complexity of miRNAs-mediated regulation. This is the first study that combines a phenotypic screening approach with RNA sequencing and bioinformatics to systematically identify miRNA-mediated pathways and to detect potential drug candidates to positively influence the phenotypic switch of VSMCs.

Highlights

In response to vascular injury vascular smooth muscle cells (VSMCs) alternate between a differentiated and a dedifferentiated state or phenotype
A recent study shows that fibroblast growth factor 12 (FGF12) reduces cell proliferation through the p53 pathway and up-regulates key factors related to the differentiation of the VSMC lineage, such as myocardin and serum response factors[10]
Pan et al.[17] found that miR-663a was significantly downregulated in VSMCs after Platelet-Derived Growth Factor (PDGF) treatment, whereas its expression markedly increased during cell differentiation

Summary

Introduction

In response to vascular injury vascular smooth muscle cells (VSMCs) alternate between a differentiated (contractile) and a dedifferentiated (synthetic) state or phenotype. Parts of the signaling cascade regulating the phenotypic switch have been described, the role of miRNAs is still incompletely understood To systematically address this issue, we have established a microscopy-based quantitative assay and identified 23 miRNAs that induced contractile phenotypes when overexpressed. The information on drug candidates was integrated into the network to predict novel combinatorial therapies that encompass the complexity of miRNAs-mediated regulation This is the first study that combines a phenotypic screening approach with RNA sequencing and bioinformatics to systematically identify miRNA-mediated pathways and to detect potential drug candidates to positively influence the phenotypic switch of VSMCs. Cardiovascular disease is a leading cause of death worldwide and accounts for more than 17.3 million deaths per year, with an estimated increase in incidence to 23.6 million by 20301. Yang et al applied in a wire-injury mouse model locally of AgomiR-22 or miR-22 inhibitor to demonstrate the modulation of the switch phenotype[23]

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