DNA methylation-mediated regulation of microRNA-140-5p affects vascular smooth muscle cell function by targeting insulin-like growth factor 1 receptor

Arteriosclerosis obliterans (ASO) is a vascular occlusive disease characterized by pathological vascular remodeling, in which the aberrant behavior of vascular smooth muscle cells (VSMCs) plays a central role. The specific role of microRNA-140-5p (miR-140-5p) in ASO remains unclear.

Vascular tissue samples from ASO patients (n = 15) and healthy controls (n = 10) were analyzed. Expression of miR-140-5p and Insulin-like Growth Factor 1 Receptor (IGF1R) was measured by quantitative real-time PCR (qRT-PCR) and Western blot. The methylation status of the MIR140 promoter was assessed by bisulfite sequencing PCR (BSP). Functional assays including EdU, Transwell, and flow cytometry were performed on cultured human VSMCs transfected with miR-140-5p mimic or inhibitor. A dual-luciferase reporter assay was used to verify the targeting relationship. An in vivo rat carotid artery balloon injury model was established.

miR-140-5p expression was significantly downregulated in ASO tissues compared to controls, associated with hypermethylation of the MIR140 gene promoter. In vitro, overexpression of miR-140-5p inhibited VSMC proliferation and migration while promoting apoptosis. IGF1R was identified as a direct target gene of miR-140-5p, and its expression was upregulated in ASO tissues, showing an inverse correlation with miR-140-5p. Immunofluorescence double staining confirmed that IGF1R co-localized with α-SMA, a marker of VSMCs. To confirm functional mediation, we performed rescue experiments using an IGF1R expression construct lacking the 3'-UTR, which renders it resistant to miR-140-5p-mediated suppression. Forced expression of this construct counteracted the inhibitory effects of miR-140-5p on VSMC proliferation and migration, as well as reversed miR-140-5p-induced apoptosis, confirming that IGF1R is a key downstream effector. In the rat model, local delivery of a miR-140-5p agomir significantly attenuated neointimal hyperplasia following arterial injury.

Promoter hypermethylation leads to silencing of miR-140-5p in ASO. Downregulation of miR-140-5p results in elevated IGF1R expression, which subsequently promotes VSMC dysfunction, contributing to ASO pathogenesis. These findings highlight the miR-140-5p/IGF1R axis as a potential therapeutic target, although the pro-apoptotic effect of miR-140-5p warrants careful evaluation of vascular integrity and safety in future translational studies.