MicroRNA-31-5p is upregulated during neointima formation and influences human coronary artery smooth muscle cell functions by regulating PIAS3 and DKK1

Abstract

Abstract Funding Acknowledgements None. Background and purpose MicroRNAs (miRs) have emerged as potential therapeutic targets in cardiovascular disease, given their ability to modulate multiple signaling pathways and cellular processes. Neointima formation as an angioplasty-related complication remains a major issue calling for innovative therapeutic options and is predominantly mediated by phenotypic and functional changes of vascular smooth muscle cells. Here, we propose that targeting miR-31-5p might be an effective approach to selectively modulate smooth muscle cell functions to enhance vascular healing and regeneration. Methods C57J/BlL6 mice (n=6, 3 months, male) were subjected to wire-induced femoral artery injury. At days 10 and 21 after injury, femoral arteries were harvested for miRNA analysis. Expression levels of miR-31-5p were confirmed by qRT-PCR in human coronary artery endothelial cells (HCAEC) and -smooth muscle cells (HCASMC). Further, the effect of miR-31-5p on cellular functions was investigated on HCAECs and HCASMCs in vitro, including migration (scratch assay), proliferation (BrdU assay), and apoptosis. Possible targets of miR-31-5p were identified in silico by structured literature research and target binding prediction and confirmed on mRNA and protein levels. High-resolution microscopy analysis was used to investigate morphological changes. Results Expression analysis revealed a significant upregulation of miR-31-5p (p<0.0001) in murine femoral artery neointimal tissue at 7 and 21 days. In vitro, miR-31-5p was significantly upregulated in HCASMC (p<0,05) but not HCAEC following serum stimulation. On a functional level, miR-31-5p revealed dissenting effects on HCAEC and HCASMC. Whereas migration and proliferation are not altered in HCAECs, knockdown of miR-31-5p significantly reduced migration (p<0,01), proliferation (p<0,05), and increased apoptosis (p<0,05) in HCASMCs. Additionally, the knockdown of miR-31-5p influenced the phenotypical switch of HCASMC towards a contractile phenotype. Under these conditions, the release of cytokines like TNF-alpha and IL1-beta was also significantly reduced (p<0,05) in HCASMCs. Systematic in silico target screens suggested DKK1 and PIAS3 as potential direct targets. Indeed, knockdown of miR-31-5p resulted in a significant upregulation of DKK1 in HCASMC (p<0,05) on mRNA level and DKK1 (p<0,05) and PIAS3 (p<0,05) on mRNA and protein level. An overexpression resulted in a significant downregulation of DKK1 in HCASMC (p<0,05) on mRNA level. Conclusion In conclusion, we report that miR-31-5p is robustly upregulated during neointima formation and seems to exert a functional neointima formation-promoting role in HCASMC rather than HCAEC. Mechanistically, DKK1 and PIAS3 as direct targets of miR-31-5p can mediate the observed effects. Thus, miR-31-5p might represent an attractive target to selectively modulate HCASMC dysfunction following vascular intervention, limiting neointima formation in treated vessels.