Regulatory Role of miR-127-3p in mTOR and AXL Pathways: Implications for Vascular Remodeling in Non-Senescent and Senescent HCASMCs

The RTK-PI3K-Akt axis plays a crucial role in regulating the functions of vascular smooth muscle cells (VSMC) by serving as a central mediator of downstream pathways, including the mTOR and NF-κB pathway, and contributing to the progression of vascular remodeling. In contrast, Micro RNAs (miRs) display a regulatory role in cellular pathways, thereby contributing to disease progression. Therefore, this study focuses on elucidating the role of miR-127-3p in non-senescent and senescent human coronary artery smooth muscle cells (HCASMC) on the mTOR and NF-κB pathway to reveal insights into vascular remodeling.

Utilizing reverse transcription and qRT-PCR we checked expression levels of miR-127-3p in human cells and murine probes. Through pre-miR transfection followed by qRT-PCR, Western blot, functional assays, and fluorescence microscopy, we examined the impact of miR-127-3p overexpression on the cells.

Upregulation of miR-127-3p expression was observed in femoral artery tissue on day 10 after wire-induced injury in C57BL/6J mice, serving as a restenosis model (p<0.001). In vitro, expression in HCASMCs is higher compared to other human cells, including endothelial cells (p<0.05), and fibroblast (p<0.05). Also, replicative senescent HCASMCs exhibit increased miR-127-3p expression (p<0.05). Expression of miR-127-3p was further increased after IFNƴ stimulation in non-senescent HCASMCs (p<0.05). MTor and AXL were identified as a target of miR-127-3p on mRNA and protein level. On mRNA level mTor was downregulated in non-senescent and replicative senescent HCASMCs (p<0.05). AXL was upregulated on mRNA and protein levels in non-senescent HCASMCs (p<0.05), whereas there was no regulation in senescent HCASMCs. In line with this, a distinct regulation of cellular function was observable for (non-) senescent cells. MiR-127-3p overexpression reduces proliferation in replicative senescent (p<0.001) and non-senescent (p<0.01) HCASMCs. However, it hinders migration in non-senescent HCASMCs (p<0.0001) but has a reverse effect in replicative senescent HCASMCs (p<0.01). Production of reactive oxygen species is increased in non-senescent HCASMCs after overexpression of miR-127-3p (p<0.5), whereas there was no effect on replicative senescent HCASMCs. LaminB1 showed a decreased expression after overexpression of miR-127-3p on mRNA level (p<0.05) in non-senescent and replicative senescent HCASMCs. In non-senescent cells, IL6 (p<0.05), p21 (p<0.05), and IL-1ß (p<0.05) showed an increased expression. These regulations were not detected in senescent HCASMCs.

The study provides compelling evidence that miR-127-3p targets mTor and AXL and thereby regulates cellular functions of non-senescent and senescent HCASMCs, contributing to the clarification of the pathogenesis underlying mechanisms. The results indicate that miR-127-3p is a key contributor to the progression of vascular diseases and suggest that targeting miR-127-3p could be an effective approach for counteracting vascular remodeling. However, the study also emphasizes the need for further research to validate miR-127-3p as a potential therapeutic target for vascular remodeling in both in vitro and in vivo settings.