Necroptosis triggered by HO is hypothesized to be a critical factor in the rupture of atherosclerotic plaques, which may precipitate acute cardiovascular events. Nevertheless, the specific regulatory molecules of this development remain unclear. We aims to elucidate a mechanism from the perspective of circular RNA. There are few studies on circRNA in VSMCs necroptosis. The objective of our research is to shed light on the intricate roles that circHIPK3 plays in the process of necroptosis in VSMCs and the development of atherosclerotic plaques that are prone to rupture. Our study elucidates the specific molecular mechanisms by which circHIPK3 regulates necroptosis and atherosclerotic vulnerable plaque formation through targeted proteins. Identifying this mechanism at the cellular level offers a molecular framework for understanding plaque progression and stability regulation, as well as a potential biomarker for the prognosis of susceptible atherosclerotic plaques. We collected clinical vascular tissue for HE staining and Masson staining to determine the presence and stability of plaques. Then, NCBI database was used to screen out circRNA with elevated expression level in plaque tissue, and the up-regulated circRNA, circHIPK3, was verified by qRT-PCR and FISH. Further, we synthesized circHIPK3′s small interference sequence and overexpressed plasmid in vitro, and verified its regulation effect on necroptosis of VSMCs under physiological and pathological conditions by WB, qRT-PCR and PI staining. Through RNA pull down, mass spectrometry and RNA immunoprecipitation, DRP1 was identified as circHIPK3 binding protein and was positively regulated by circHIPK3. Meanwhile, on the basis of silencing of DRP1, the regulation of circHIPK3 on necroptosis is verified to be mediated by DRP1. Finally, we validated the regulation of circHIPK3 on vulnerable plaque formation in ApoE mice. We investigated that circHIPK3 was highly expressed in vulnerable plaques, and the increase in expression level promoted HO induced necroptosis of VSMCs. CircHIPK3 targeted the protein DRP1, leading to an elevation in mitochondrial division rate, resulting in increased reactive oxygen species and impaired mitochondrial function, ultimately leading to necroptosis of VSMCs and vulnerable plaque formation. CircHIPK3 interact with DRP1 involve in HO induced Mitochondrial damage and necroptosis of VSMCs, and Silencing circHIPK3 in vivo can reduce atherosclerotic vulnerable plaque formation. Our research findings may have applications in providing diagnostic biomarkers for vulnerable plaques.

中文翻译：

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CircHIPK3靶向DRP1介导过氧化氢诱导的血管平滑肌细胞坏死性凋亡和动脉粥样硬化易损斑块形成

据推测，H2O 引发的坏死性凋亡是动脉粥样硬化斑块破裂的关键因素，可能会引发急性心血管事件。然而，这种发展的具体调控分子仍不清楚。我们的目标是从环状RNA的角度阐明其机制。目前关于 circRNA 在 VSMC 坏死性凋亡中的研究较少。我们研究的目的是阐明 circHIPK3 在 VSMC 坏死性凋亡和易于破裂的动脉粥样硬化斑块发展过程中所发挥的复杂作用。我们的研究阐明了 circHIPK3 通过靶向蛋白调节坏死性凋亡和动脉粥样硬化易损斑块形成的具体分子机制。在细胞水平上识别这种机制为理解斑块进展和稳定性调节提供了分子框架，并为易受影响的动脉粥样硬化斑块的预后提供了潜在的生物标志物。我们收集临床血管组织进行HE染色和Masson染色以确定斑块的存在和稳定性。然后利用NCBI数据库筛选出斑块组织中表达量升高的circRNA，并通过qRT-PCR和FISH对上调的circRNA circHIPK3进行验证。进一步，我们在体外合成了circHIPK3的小干扰序列和过表达质粒，并通过WB、qRT-PCR和PI染色验证了其对生理和病理条件下VSMCs坏死性凋亡的调节作用。通过RNA Pull down、质谱和RNA免疫沉淀，DRP1被鉴定为circHIPK3结合蛋白，并受到circHIPK3的正向调控。同时，在DRP1沉默的基础上，验证了circHIPK3对坏死性凋亡的调节是由DRP1介导的。最后，我们验证了 circHIPK3 对 ApoE 小鼠易损斑块形成的调节作用。我们研究发现circHIPK3在易损斑块中高表达，表达水平的增加促进H2O诱导VSMCs坏死性凋亡。 CircHIPK3靶向蛋白质DRP1，导致线粒体分裂率升高，导致活性氧增加和线粒体功能受损，最终导致VSMC坏死和易损斑块形成。 CircHIPK3与DRP1相互作用参与H2O2诱导的线粒体损伤和VSMCs坏死性凋亡，体内沉默circHIPK3可以减少动脉粥样硬化易损斑块的形成。我们的研究结果可能应用于为易损斑块提供诊断生物标志物。