低氧下调miR-223在肺动脉高压形成中的作用及分子机制研究

Pulmonary arterial hypertension (PAH) is a complex vascular disease with a dreadful survival rate. Hypoxia-induced pulmonary arterial hypertension (HPH) is characterized by abnormal proliferation of pulmonary artery smooth muscle cell (PASMC) and progressive structural remodeling of pulmonary arteries. Our preliminary data showed that miR-223 was remarkably down-regulated in hypoxia-treated PASMC and lung tissues, and RhoB, a member of Rho/Rho kinase pathway, was significantly up-regulated by hypoxia. Further studies confirmed that RhoB is a direct target of miR-223. Functionally, miR-223 inhibits the proliferation of PAMSC. Based on the results above, we propose that the development of HPH might be associated by miR-223 through the regulation of Rho/Rho kinase pathway. In this proposal, the targeting of Rho/Rho kinase pathway by miR-223, as well as its influence upon proliferation and migration of PASMC will be explored. Using hypoxia induced HPH rat model, the impact of synthetic miR-223 sprays on pulmonary vascular remodeling and HPH symptoms will be investigated. Finally, the clinical relationship between miR-223 level and PAH disease will be evaluated. The purpose of this project is to elucidate the molecular mechanism of HPH regulated by miR-223 via Rho/Rho kinase pathway and might provide a new clue for the development of miRNA-based treatment and early diagnostics means for HPH.

肺动脉高压的发病机制复杂，死亡率高。多认为肺动脉平滑肌细胞(PASMC)异常增殖和肺血管结构重建是低氧性肺动脉高压(HPH)的重要特征。前期研究表明，低氧使miR-223在PASMC和小鼠肺组织表达下调，而RhoB蛋白则上调；且RhoB是miR-223的靶基因；在功能上，miR-223抑制PASMC增殖。本项目推测，miR-223通过RhoB参与的Rho/Rho激酶信号通路调控HPH的发生。为了验证该假设，拟从细胞水平研究miR-223对Rho/Rho激酶通路的调控，及在此基础上引起的PASMC增殖、迁移等变化；以HPH大鼠为模型，探讨miR-223喷剂对HPH症状的改善作用；最后通过PAH患者样本分析，明确miR-223与PAH在临床上的关系。本项目不仅有助于从miRNA调控Rho/Rho激酶通路的角度阐明HPH的分子机理，还能为开发基于miRNA的治疗和早期诊断手段提供新思路。

背景：慢性缺氧是肺动脉高压（PAH）形成的主要原因，缺氧诱导基因异常表达，引起肺动脉平滑肌细胞（PASMC）异常增殖和迁移，从而导致肺动脉血管重构和肺动脉高压的形成。microRNA（miRNA）是一类非编码单链小RNA，通过与靶基因3`非编码区相互作用，在转录后水平抑制目的基因的表达。研究发现miRNA与肺动脉高压的形成有关，但潜在的机制仍不十分清楚。.方法和结果：在本研究中，我们发现miR-223在低氧诱导的小鼠和大鼠的肺组织、肺动脉，以及低氧诱导的肺动脉平滑肌细胞（PASMC）中表达量均显著下调。在细胞水平上，抑制miR-223的表达能促进PASMC增殖，而过表达miR-223则可以抑制PASMC的增殖、迁移和应力纤维的形成。在动物水平上，导入miR-223 agomir能抑制低氧诱导小鼠的肺动脉压升高和远端肺小动脉的肌化。我们证明RhoB是miR-223的靶基因，其在低氧诱导下表达量上调。挽救实验证实，过表达RhoB可以逆转miR-223的过表达对PASMC增殖的抑制作用。过表达miR-223能够抑制RhoB的表达并抑制下游MYPT1的磷酸化和MLC2的表达，MLC2随后也被证实是受miR-223直接调控的靶基因。此外，临床样本分析发现，miR-223在女性先天性心脏病合并PAH(CHD-PAH)患者的血清中表达显著降低，而在男性患者中没有明显差异。.结论：我们的研究结果首次证明了mir-223通过靶基因RhoB和MLC2调节PASMC的增殖、迁移和肌动球蛋白重组，从而影响肺动脉血管重构和肺动脉高压的发展。同时提示，miR-223可能作为一种潜在的生物标志物和小分子药物应用于PAH的诊断和治疗。