MiR-375 Targeting PDK1 Regulates the Wnt/β-Catenin Pathway in Lower Extremity Arteriosclerosis Obliterans

Zongrong Liu,1 Zhujun Yue,1 Ruirui Yang,1 Safwan Muhammad,1 Jingquan Chen21Graduate School, North Sichuan Medical College, Nanchong, Sichuan, People’s Republic of China; 2Department of Vascular Surgery, Affiliated Hospital of North Sichuan Medical College, Nanchong, Sichuan, People’s Republic of ChinaCorrespondence: Jingquan Chen, Department of Vascular Surgery, Affiliated Hospital of North Sichuan Medical College, Nanchong, Sichuan, People’s Republic of China, Tel +8615196641235, Email [email protected]: Lower extremity arteriosclerosis obliterans (LEASO) is a severe peripheral arterial disease characterized by chronic inflammation, lipid deposition, and vascular remodeling. MicroRNA-375 (miR-375) has emerged as a regulatory molecule in vascular pathology, but its role in LEASO remains unclear.Objective: This study investigated whether miR-375 regulates the Wnt/β-catenin signaling pathway by targeting pyruvate dehydrogenase kinase 1 (PDK1), and its impact on LEASO progression.Methods: Clinical samples (arterial tissues and blood) were collected from 10 LEASO patients and 10 trauma-related amputation controls. Histology, qPCR, Western blot, and ELISA were used to evaluate molecular and inflammatory markers. Animal models of atherosclerosis (LDLR-/- mice) and LEASO (SD rats) were established, with Wnt/β-catenin pathway inhibition as intervention. In vitro, vascular smooth muscle cells (VSMCs) were transfected with miR-375 mimics/inhibitors, and functional assays were performed. Dual-luciferase reporter assays confirmed the interaction between miR-375 and PDK1.Results: Compared with the control group, miR-375 expression was markedly downregulated in LEASO tissues, reaching only 50.3% of the control level (P=0.008). By contrast, the mRNA and protein expression levels of PDK1, Wnt3a and β-catenin were significantly increased. A strong positive correlation was observed between PDK1 expression and Wnt signaling activity (r=0.91, P=0.012). Dual-luciferase reporter assay verified that miR-375 directly bound to the 3′-untranslated region (3′-UTR) of PDK1 mRNA, and suppressed PDK1 expression by 78% (P< 0.001).In vascular smooth muscle cells (VSMCs), miR-375 overexpression suppressed cell proliferation and migration by 41.5% and 17.3%, respectively. Nevertheless, simultaneous PDK1 overexpression reversed these inhibitory biological effects. Notably, miR-375 upregulation inhibited the PDK1-Wnt/β-catenin signaling cascade and alleviated pathological phenotypes both in vitro and in vivo. However, endogenous miR-375 reduction was accompanied by compensatory and nonlinear alterations of molecular profiles under certain in vitro conditions, such as oxidized low-density lipoprotein (ox-LDL)-stimulated VSMCs.In vivo experiments demonstrated that restored miR-375 expression reduced atherosclerotic plaque area by 41.5%, decreased the secretion of pro-inflammatory cytokines including interleukin-6 (IL-6) and interleukin-1β (IL-1β), and enhanced plaque stability. Conversely, miR-375 knockdown aggravated intracellular lipid deposition and systemic inflammatory responses.Conclusion: miR-375 suppresses LEASO progression by targeting PDK1 and downregulating Wnt/β-catenin signaling. Restoring miR-375 expression represents a promising therapeutic strategy for LEASO.Keywords: lower extremity arteriosclerosis obliterans, miR-375, PDK1, Wnt/β-catenin, vascular smooth muscle cells, inflammation, targeted therapy