血管特异性长链非编码RNA RASAL2-AS1在调控血管平滑肌细胞表型转换中的作用和机制研究

Vascular remodeling is one of the major causes of the complications of hypertension. VSMCs phenotypic switching plays a central role during the development of vascular remodeling while the mechanisms remain unclear. Long non-coding RNAs have gained prominence over the last couple of years as fundamental regulators of cellular functions, and their dysregulation is increasingly being linked to pathology. Using lncRNA-microarray, we screened out a vascular tissue-specific lncRNA, RASAL2-AS1, whose expression level increases significantly in remodelled vascular. In vitro, we found RASAL2-AS1 promote VSMCs phenotypic switching. We then compared the transcriptome before and after RASAL2-AS1 knock-down, finding that RASAL2-AS1 promotes transcription of AT1R, which is a key regulator of VSMCs phenotypic switching. Our former work showed that HDAC1 represses transcription of AT1R and SUMOylation is one of the main post translational modifications of HDAC1. Through RIP assay, we confirmed the direct connection between lncRNA RASAL2-AS1 and SUMO-1 protein. We also found the SUMOylation of HDAC1 decreased after RASAL2-AS1 knock-down in VSMC. Therefore, we suspect that the over-expressed lncRNA RASAL2-AS1 connects with SUMO-1 protein directly, leading to the decrease of HADC1 SUMOylation, which in turn decreases the transcription repression of AT1R, and resulting in VSMCs phenotypic switching. Our study will confirm the role of RASAL2-AS1 in VSMCs phenotypic switching in vivo, in vitro and in transgenic/gene knock-out animals, testing the hypothesis that RASAL2-AS1 participates in the regulation of VSMCs phenotypic switching, which will provide potential therapeutic targets for vascular remodeling.

VSMCs表型转换在血管重构中发挥关键作用，但机制尚不清楚。我们前期实验发现血管特异性lncRNA RASAL2-AS1在重构血管较正常血管表达显著升高，抑制RASAL2-AS1有助于VSMCs收缩表型的维持，进一步发现RASAL2-AS1可能通过促进AT1R转录参与VSMCs表型转换。已有研究表明HDAC1能够抑制AT1R转录，SUMO-1介导的苏木化是HDAC1重要修饰方式。我们发现RASAL2-AS1能够与SUMO-1蛋白直接结合，降低HDAC1苏木化水平。因此我们提出假说：RASAL2-AS1通过竞争性结合SUMO-1蛋白，抑制SUMO-1介导的HDAC1苏木化，导致HDAC1活性下降，从而降低HDAC1对AT1R的转录抑制，促进VSMCs表型转换。本课题将构建转基因及基因敲除动物模型，进一步研究RASAL2-AS1促进VSMCs表型转换机制，为血管重构提供新的治疗靶点。

血管平滑肌细胞（VSMCs）表型转换在血管重构中发挥关键作用，但机制尚不清楚。我们发现通过基因芯片筛选发现了一个新的血管特异性表达的lncRNA RASAL2-AS1，其在在重构血管较正常血管表达显著升高，RNA 荧光原位杂交显示，RASAL2-AS1 主要分布于血管中膜VSMCs 。敲降VSMCs 中RASAL2-AS1 后，反映VSMC收缩态表型的生物标志分子，包括α-Actin、Calponin 表达显著上升；VSMCs 迁移、增殖能力显著下降；LncRNA RASAL2-AS1基因敲除大鼠颈动脉球囊损伤后VSMCs增殖显著低于野生型大鼠，说明抑制RASAL2-AS1 有助于VSMCs 收缩表型的维持。我们通过表达谱芯片比较了VSMCs 中RASAL2-AS1 敲降后转录组的变化，并通过Q-PCR 进一步验证发现血管紧张素Ⅱ1 型受体（AT1R）的转录随着RASAL2-AS1 受到抑制而显著下降，提示RASAL2-AS1 通过调控AT1R 参与VSMCs 表型转换。AT1R在转录水平受组蛋白去乙酰化酶（HDAC1）的调控，HDAC1可以作用于AT1R启动子，抑制AT1R转录，苏木化是HDAC1重要修饰方式。生物信息学(catRAPID)预测RASAL2-AS1与SUMO1可能直接结合，我们进一步通过RIP及Q-PCR证实了VSMCs中RASAL2-AS1可以竞争性结合SUMO1，抑制SUMO-1介导的HDAC1苏木化，导致HDAC1活性下降，从而降低HDAC1对AT1R的转录抑制，促进VSMCs表型转换。本研究发现lncRNA RASAL2-AS1 参与VSMCs 表型转换的新机制，为血管重构提供新的治疗靶点。