MicroRNA-31-5p is upregulated during neointima formation and significantly impacts human coronary artery smooth muscle cell functions through its interaction with STK40

Background and purpose: MicroRNAs (miRs) have been identified as potential therapeutic targets in cardiovascular disease due to their ability to influence multiple signaling pathways and cellular processes. Neointima formation, which is a major complication associated with angioplasty, is primarily caused by changes in the behavior and function of vascular smooth muscle cells. Therefore, we suggest that targeting miR-31-5p could be an effective way to specifically modify smooth muscle cell functions and promote vascular healing and regeneration.

Methods: C57BL/6J mice (6 male, 3 months old) underwent wire-induced femoral artery injury. The femoral arteries were collected for miRNA analysis on days 7 and 21 post-injury. Quantitative real-time PCR (QRT-PCR) was used to confirm the expression of miR-31-5p in human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells (HCASMC). In vitro studies were conducted to investigate the effects of miR-31-5p on cellular functions, including migration (scratch assay), proliferation (BrdU assay), and apoptosis. Potential targets of miR-31-5p were identified through structured research on RNA binding prediction and RNA sequencing. The functional role of the identified targets was further investigated in vascular cells.

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. An RNA sequencing approach identified STK40, LATS2, and GXYLT1 as targets of miR-31-5p regulation in HCASMC. Their regulation was confirmed on mRNA and protein levels. Transfection with anti-miR-31-5p resulted in a significant downregulation of STK40 and LATS2 (p<0.01 and p<0.05). Further investigations focused on the role of STK40, a kinase that does not affect cellular proliferation and migration in HCASMC. However, siRNA transfection for STK40 significantly reduces apoptosis (p<0.05). Additional experiments confirmed that STK40 regulation is important for anti-miR-31-5p-induced apoptosis in HCASMC (p<0.05).

Conclusion: In conclusion, our findings demonstrate the robust upregulation of miR-31-5p during neointima formation and its significant role in promoting neointima formation in HCASMC rather than HCAEC. Mechanistically, we have identified STK40 as a direct target of miR-31-5p, mediating, among other functions, the observed apoptotic effect in HCASMC. Therefore, miR-31-5p presents a compelling target for selectively modulating HCASMC dysfunction following vascular intervention, thus potentially limiting neointima formation in treated vessels.