MiR-32-5p as a potential key mediator of vascular remodeling: implications for enhancing vascular smooth muscle cell proliferation and morphological alterations via PIK3R3 inhibition

Introduction: Dysfunctional endothelial cells (EC) and smooth muscle cells (SMC) play a pivotal role in vascular remodeling. The enhancement of inflammation, oxidation and vasoconstriction, subsequently results in pathological changes of the vessel wall and therefore critically influencing the progression of cardiovascular diseases. Especially the accumulation of senescent SMC amplifies these processes. Recent studies identified microRNAs (miRNAs) as key regulators of cellular function, highlighting their potential as therapeutic targets for vascular remodeling. This study investigates the role of miR-32-5p in SMC function and its potential as a therapeutic target.

Material and Methods: Experiments were conducted in (non-) senescent human coronary artery smooth muscle cells (HCASMC). Initially, miR-32-5p expression levels were analyzed in both ECs and HCASMC using quantitative RT-PCR. The functional impact of miR-32-5p on migration, proliferation, and cell survival was evaluated through scratch wound and BrdU assays. Downstream targets were identified, and their expression was assessed at both mRNA and protein levels. Immunofluorescence microscopy was utilized to evaluate morphological changes.

Results: Our findings revealed that miR-32-5p expression was significantly elevated in HCASMC compared to EC, with levels decreasing in replicative senescent cells (p<0.05). Stimulation with growth factors led to an increase in miR-32-5p expression (p<0.05), indicating its role in cell proliferation. Indeed, transfection with pre-miR-32-5p significantly enhanced HCASMC proliferation (p<0.05), while reducing cell migration (p<0.05). Conversely, knockdown of miR-32-5p in senescent cells also decreased migration (p<0.05). No apoptotic effects were observed following miR-32-5p transfection. In silico analysis identified several miR-32-5p targets in HCASMC, which were experimentally confirmed on mRNA level including KLF4 (p<0.05), MAP2K4 (p<0.05), BCL2 (p<0.01), ADAM10 (p<0.05), FBXW7 (p<0.05), PIKFYVE (p<0.01), and PIK3R3 (p<0.05). Further, PIK3R3, FBXW7 and PIKFYVE were also regulated on protein level (p<0.05). Next, senescence markers were assessed to determine possible effects of miR-32-5p on cellular senescence. As a result, increased p21 and decreased Lamin B1 levels (p<0.01) were detected in non-senescent cells, alongside elevated p53 (p<0.01), p14ARF, p16INK4a, and p21 in senescent cells (p<0.05). Cytokine analysis demonstrated that IL-8 levels were significantly reduced following pre-miR transfection in both (non-) senescent HCASMC (p<0.01). Immunofluorescence staining revealed that miR-32-5p overexpression induced a distinct rhomboid morphology in HCASMC.

Conclusion: These compelling findings position miR-32-5p as a critical regulator of HCASMC function, emphasizing its potential as a therapeutic target in atherosclerosis. To comprehensively clarify its role in the pathogenesis of atherosclerosis and its implications for cardiovascular therapies, further investigations are needed. Future experiments will explore downstream target interactions and include Western blot analyses to quantify phenotypic changes, as well as siRNA studies to disclose the functional pathways of miR-32-5p.