解析ROS/Oct1通路负调控miR-30b/133a1表达的分子机制及促进主动脉瓣钙化的作用

Recent studies have demonstrated that the phenotypic and functional changes of valvular interstitial cells were ongoing throughout AVC progression, and reactive oxygen species (ROS) was the original factor for AVC pathogenesis. Our preliminary findings showed that miR-30b/133a1 had the ability to regulate the phenotypic and functional changes of valvular interstitial cells, the expression of these two miRNAs were downregulated by ROS and Oct1, and Oct1 also decreased Oct1 expression. Accordingly, we proposed a hypothesis for "cascade regulation of ROS/Oct1/miR -30b-133a1" and speculated that this pathway is the promoptor for AVC progrssion. To test this hypothesis, we will take valvular interstitial cells as the carrier, and explore, step by step, the transcriptional regulation mechanism of ROS on Oct1, and Oct1 on miR-30b/133a1; based on the above findings, we will examine whether Oct1 mediates the regulation of miR-30b/133a1 by ROS; additionally, by using in vitro and in vivo model, we will investigate the role of ROS/Oct1 on valvular fibrosis and calcification. Through this research, we try to clarify the mechanisms to regulate the expression of miR-30b/133a1, and the effect of ROS/Oct1 on AVC progression, and construct a new model for "miRNA expression regulation" research.

新研究表明瓣膜间质细胞行为改变是贯穿主动脉瓣钙化(AVC)进展的中轴线，并明确活性氧簇(ROS)是AVC始发因素。前期,我们证实miR-30b/133a1是稳定瓣膜间质细胞生理行为的精密调节器，其表达受ROS和Oct1的调控，而ROS又负调控Oct1的表达,据此我们提出"ROS/Oct1/miR-30b-133a1级联调控"假说，并推测该通路是促进AVC进展的推进器。为验证这一假说，我们将以瓣膜间质细胞为载体，逐步分析ROS对Oct1、Oct1对miR-30b/133a1的转录调控作用和机制;并以此为基础，考察Oct1是否介导ROS对miR-30b/133a1的表达调控；此外，构建体内外模型，进一步明确ROS/Oct1对AVC进展的促进作用。通过此研究，力图阐明导致miR-30b/133a1表达下调的机制及ROS/Oct1对AVC进展的作用，为"miRNA自身表达调控机制"研究提供新模型。

深入研究主动脉瓣钙化的发病机制，揭示关键调控环节，已成为当今老年性疾病防治领域中亟待攻克的课题和科研热点。在本项目研究中，我们以ROS/Oct1/ miRNA30b- miRNA 133a1-Twist1通路为研究主线，明确信号轴关键节点基因对瓣膜间质细胞表型转分化及钙化的调控作用。研究结果表明：ROS氧化应激模型中瓣膜间质细胞生长抑制、凋亡细胞增多。Oct1在钙化性瓣膜中的表达显著升高，但是，远离钙化区的标本中Oct1蛋白表达显著高于钙化区标本；构建瓣膜间质细胞钙化模型，Oct1表达的最高峰是第7天，随后降低，第21天达最低峰；ROS促进Oct1的表达和蛋白核转移。在ROS氧化应激模型中，Oct1能抑制瓣膜间质细胞凋亡、及caspase-3的表达，同时促进cleaved- caspase-3 Catalase、MnSOD、ecSOD的表达；瓣膜间质细胞钙化模型中，Oct1促进瓣膜间质细胞表达ɑ-SMA，细胞收缩能力增强，并降低Runx2、OPN的表达，抑制钙盐沉积。miRNA30b在钙化性瓣膜、瓣膜间质细胞钙化模型中中的表达显著降低，并且miRNA30b的下降伴随着Runx2、osteocalcin、Smad1、caspase-3 表达升高；miRNA30bmiR-30b 直接通过结合 Runx2, Smad1 及 caspase-3 mRNA 的 3’UTR 发挥了转录后抑制作用；ROS能抑制miRNA30b、miRNA133a1的表达。Twist1在钙化性瓣膜、瓣膜间质细胞钙化模型中，Twist1的表达量显著降低；Twist1结合到Runx2 基因 P2 启动子区的E-box-820进而抑制RUNX2及其下游基因OCN、OPN的表达。以上结果表明：ROS是瓣膜间质细胞损伤的始发因素。Oct1促进瓣膜间质细胞激活、向肌纤维母细胞转分化，对抗ROS所导致的细胞损伤,并抑制瓣膜间质细胞向骨母细胞转分化，降低钙盐沉积。miRNA30b、miR133a1受ROS的表达调控，其中miRNA30b能直接靶向抑制Runx2, Smad1 及 caspase-3的表达而抑制瓣膜间质细胞向骨母细胞转分化，降低凋亡。Oct1能靶向调控Twist1的转录表达，而Twist1又可直接转录调控Runx2的表达，抑制瓣膜间质细胞向骨母细胞转分化。