MiR-92b-3p modulates apoptosis in smooth muscle cells through BCL2L11 regulation, unravelling novel therapeutic strategies for unstable plaques

Abstract

Abstract Funding Acknowledgements None. Smooth muscle cell (SMC) apoptosis is a contributory factor to plaque rupture in atherosclerosis, thereby promoting plaque destabilization through various mechanisms such as thrombogenic activation and plaque microcalcification. Immunohistochemical analysis has revealed elevated expression of proteins associated with intrinsic apoptosis, such as BCL2L11 (Bim), in unstable atherosclerotic plaques. Our hypothesis posits that Bim is a direct target of miR-92b-3p, an understudied microRNA, prompting an investigation into its role in regulating apoptosis in SMCs. Our objective is to discern a novel therapeutic axis for preventing plaque rupture by employing miRNA-based targeted interventions. Within a model of primary human vascular cells, we employed various in vitro techniques, including PCR, Western Blot, Live cell imaging, and ELISA. Lipotransfection facilitated transient modulation of miR-92b-3p, using both knockdown and elevation, while an siRNA transfection model was also utilized. MiR-92b-3p exhibits robust expression in late atherosclerotic lesions in vivo (p<0.001) and demonstrates heightened levels in smooth muscle cells (SMCs) compared to endothelial cells (ECs) in vitro (p<0.01) and in vivo in respective tissues (p<0.01). Bim, a direct target of miR-92b-3p, experiences upregulation post-miR-92b-3p knockdown and downregulation following pre-miR-92b-3p introduction into SMCs, both at mRNA and protein levels (p<0.01, p<0.01). Apoptosis increases in growth-medium cultured SMCs after miR-92b-3p knockdown (p<0.05), but not in senescent SMCs, ECs, or basal-medium treated SMCs. Live-cell imaging reveals a reduction in SMC count after anti-miR-92b-3p transfection (p<0.01), accompanied by cell shrinkage indicative of apoptosis 72 hours post-transfection (p<0.05). Growth factor stimulation results in miR-92b-3p upregulation (p>0.05) and Bim downregulation in SMCs. SiRNA knockdown of BIM in SMCs abolishes apoptosis induction through miR-92b-3p knockdown (p<0.05). Conversely, in apoptotic SMCs, miR-92b-3p upregulation protects cells from apoptosis (p<0.05). MiR-92b-3p effectively modulates mitochondrial apoptosis in SMCs by targeting BIM. This regulatory impact is exclusive to proliferative SMCs, known for their deleterious involvement in atherosclerotic plaques. Cumulatively, the elevation of miR-92b-3p via targeted miRNA therapeutics or miRNA-drug interactions in the advanced stages of atherosclerosis presents a potential therapeutic avenue for averting plaque rupture.