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

Simon Gürlach (Halle (Saale))1, K. Kalies (Halle (Saale))1, F. Daniel (Halle (Saale))1, S. Böttcher (Halle (Saale))1, J. Köster (Halle (Saale))1, L. Hehl (Halle (Saale))1, K. Knöpp (Halle (Saale))1, J. M. Daniel (Halle (Saale))1, D. G. Sedding (Halle (Saale))1

1Universitätsklinikum Halle (Saale) Klinik und Poliklinik für Innere Medizin III Halle (Saale), Deutschland


In the context of cardiovascular diseases (CVDs), microRNAs (miRs) have emerged as potential therapeutic targets, given their ability to modulate multiple signaling pathways and cellular processes. Further, neointima formation as an angioplasty-related complication remains a major issue calling for innovative therapeutic options. Vascular smooth muscle cells and their phenotypic and functional changes play a crucial role in vascular homeostasis and pathological processes such as the development of neointimal tissue after vascular injury. Here, we propose that targeting miR-31-5p might be an effective approach to selectively modulate smooth muscle cell functions in order to enhance vascular healing and regeneration.

An initial screening of murine neointimal tissue for differentially regulated miRs revealed robustly increased expression levels of miR-31-5p over time. Subsequently, expression levels of miR-31-5p were confirmed by quantitative reverse transcription polymerase chain reaction (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. 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.

Expression analysis revealed a significant upregulation of miR-31-5p (p<0.0001) in C57BL/6J mice following wire-induced femoral artery injury 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 influences the phenotypical switch of HCASMC towards a contractile phenotype. Under these conditions, the release of cytokines like TNF-alpha and IL1-beta is also significantly reduced (p<0,05) in HCASMCs. Systematic in silico target screens suggested DKK1 and PIAS3 as potential targets. 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.

In conclusion, we report that miR-31-5p is robustly upregulated during neointima formation and seems to exert a functional role in HCASMC rather than HCAEC since knockdown of miR-31-5p was able to prevent HCASMC proliferation and migration and induce apoptosis. Mechanistically, DKK1 and PIAS3, identified as direct targets of miR-31-5p and 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.

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