miR-124调节TNFα介导平滑肌细胞胶原合成酶P4Hα1表达机制及对斑块易损性的作用

Collagen plays an important role in plaque stabilization. Inflammation is one of the main modulators of collagen metabolism. However, studies of inflammation-induced plaque destabilization were mainly focused on collagen degradation. Little is known about the role the other part of collagen metabolism, collagen synthesis, during the process. P4Hα1 is a key enzyme during synthesis of collagens. Our preliminary experiments showed that in destabilized plaques, P4Hα1, together with Collagen I and III, was significantly suppressed, whereas miR-124 was obviously upregulated. We preconceived that miR-124 suppress P4Hα1 and subsequently Collagen I and III to destabilize plaques with the presence of inflammation factors, such as TNFα. We will investigate the mechanisms involved in miR-124-mediated modulation of collagen synthesis and plaque stability by TNFα both in vitro and in vivo.RT-RCR,Western Blot，transfection of lenti-virus, luciferase reporter gene assay, EMSA, CHIP, miRNA detection and analysis techniques will be applied. The study will include molecular and cellular experiments, organic experiments and animal experiments. We hope that our research will contribute to the understanding of the role of collagen synthesis in plaque destabilization and provide new insight into prevention and treatment of vulnerable plaques and acute cardiovascular events.

斑块炎症、胶原纤维含量与斑块易损性有关，P4Hα1是胶原合成过程的关键酶。炎症因子对胶原合成和斑块稳定性研究甚少，且机制不明确。我们研究发现在易损斑块中miR-124表达显著升高，P4Hα1表达降低；TNFα抑制P4Hα1的表达，P4Hα1过表达可增加胶原含量和斑块稳定性，但其作用机制尚需研究。为此我们提出假说：TNFα经NF-Y/FOXP1增加miR-124表达，miR-124可通过降低P4Hα1蛋白表达，降低斑块胶原蛋白含量而增加斑块易损性。本课题将从体内外研究验证这一假说，利用RT-PCR、Western blot、慢病毒载体转染、荧光素酶报告基因、融合蛋白、EMSA、CHIP及miRNA等技术，从分子、细胞、组织以及动物整体水平等探讨miR-124在炎症影响胶原合成和斑块稳定性过程中的作用和分子机制。本研究将从胶原合成角度揭示易损斑块发生机制，为稳定斑块的干预策略提供新的思路。

斑块炎症、胶原纤维含量与斑块易损性密切相关，但目前炎症因子对胶原合成和斑块稳定性研究甚少，且机制不明确。据报道，miR-124与多种胶原代谢疾病密切相关，但其与动脉粥样硬化的关系尚不明确。经动物实验发现，在炎症刺激过程中ApoE-/-小鼠动脉粥样硬化斑块内miR-124含量显著升高，我们对miR-124在血管平滑肌细胞中与炎症因子的关系进行了观察，发现TNFα、IL-6等炎症因子不能稳定调节miR-124的表达，经筛查，IL-1β对miR-124有稳定的上调作用，同时其对胶原有明显的抑制作用。研究发现miR-124可直接靶向作用于胶原合成限速酶P4Hα1的mRNA，抑制其转录后翻译过程，使其表达减少，从而抑制胶原合成。研究证明IL-1β可经miR-124- P4Hα1轴对胶原代谢产生影响，此外，转录因子c-Rel可能参与了IL-1β对miR-124的调节过程。我们经体内实验验证，miR-124可使P4Hα1表达下降，使动脉粥样硬化斑块内胶原含量减少，从而降低斑块稳定性。我们的研究揭示了炎症因子IL-1β通过c-Rel-miR-124-P4Hα1-胶原轴调节动脉粥样硬化发展过程的新机制，为稳定斑块的干预策略提供了新的思路。