Background and purpose: Neointima formation, a common complication of angioplasty, primarily arises from vascular smooth muscle cell (HCASMC) dysfunction and inflammation, leading to pathological vascular remodeling. MicroRNAs (miRs) have emerged as promising therapeutic targets in cardiovascular disease, given their capacity to regulate multiple signaling pathways and cellular functions. This study suggests targeting miR-31-5p as an effective approach to selectively 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 miR-31-5p expression 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 properties of the identified targets were further investigated in vascular cells. Immunofluorescence microscopy of murine aortic tissue was performed. Results: Expression analysis revealed a significant upregulation of miR-31-5p (p<0.0001) in murine femoral artery neointimal tissue at days 7 and 21 post-injury. In vitro, miR-31-5p expression was significantly increased in HCASMCs (p<0.05), but not in HCAECs, following serum stimulation. Functionally, miR-31-5p exerted distinct effects in the two cell types. While migration and proliferation remained unchanged in HCAECs, knockdown of miR-31-5p in HCASMCs significantly reduced migration (p<0.01) and proliferation (p<0.05), and increased apoptosis (p<0.05). Under these conditions, RNA levels of TNF-α and IL1-β were also significantly reduced (p<0.05). RNA sequencing identified STK40, LATS2, and GXYLT1 as potential targets of miR-31-5p in HCASMCs, which were validated at mRNA and protein levels. Transfection with anti–miR-31-5p led to significant downregulation of STK40 (p<0.01) and LATS2 (p<0.05), whereas these targets were not regulated in HCAECs. At the protein level, indirect targets including SMMHC, smoothelin, DKK1, PIAS3, and KLF4 were significantly upregulated following miR-31-5p knockdown. Further analyses focused on STK40, a kinase that does not affect proliferation or migration, but whose siRNA-mediated silencing markedly reduced apoptosis (p<0.05). Additional experiments confirmed that STK40 regulation is required for anti–miR-31-5p–induced apoptosis in HCASMCs (p<0.05). Immunofluorescence microscopy demonstrated co-localization of smoothelin and STK40 in murine aortic cross sections. Conclusion: In summary, miR-31-5p is strongly upregulated during neointima formation, highlighting its crucial role in this process, specifically in HCASMC rather than HCAEC. Mechanistically, we identified STK40 as a direct target of miR-31-5p, which contributes to the observed apoptotic effects in HCASMC. These insights suggest that miR-31-5p is a promising target for selectively addressing HCASMC dysfunction following vascular intervention, offering potential to reduce neointima formation in treated vessels.
