LncRNA TCONS_00059579作为ceRNA调控miR-322/apelin在高原肺动脉高压发生中的作用机制研究

The vasodilatation of apelin plays an important role in the progression of high altitude pulmonary hypertension (HAPH). But the mechanism is not clear that what causes the reduction of apelin and triggers HAPH at high altitude. The applicants have found in previous studies that there was a correlation between TCONS_00059579, miR-322 and apelin，the preliminary inhibition experiment of miR-322 confirmed that with the reduction of miR-322, the expression of TCONS_00059579 and apelin got a corresponding increase. This trend indicates that TCONS_00059579 may inhibit the expression of apelin by competitive inhibition of miR-322, which matches well with the competing endogenous RNA(ceRNA) hypothesis. The aim of this study is to clarify the regulatory mechanism of TCONS_00059579 in the expression of miR-322/apelin and to further illuminate the pathogenesis of HAPH which will provide a theoretical and experimental basis for the prevention and treatment of HAPH.

Apelin的血管舒张功能对高原肺动脉高压（HAPH）的发生具有重要抑制作用，而在高原低氧环境，何种原因导致apelin表达降低并引发HAPH的机理尚不明确。申请人在前期研究中发现lncRNA TCONS_00059579、miR-322、apelin之间存在着关联性，并通过miR-322抑制实验证实，miR-322降低后，TCONS_00059579、apelin表达水平升高，三种RNA水平的变化趋势与ceRNA调控理论相吻合，表明TCONS_00059579可能通过竞争性抑制miR-322实现对apelin的调控。为验证以上推论，本项目拟通过体外、体内实验，研究TCONS_00059579在miR-322/apelin表达调控中的作用机制，进一步阐明HAPH的发病机理，为防治HAPH提供理论和实验依据。

Apelin血管舒张功能对高原肺动脉高压（HAPH）的发生具有重要抑制作用，而高原缺氧环境，何种原因导致Apelin表达降低并引发HAPH的问题仍需深入解决。本课题探讨LncRNA-TCONS_00059579、miR-322、Apelin表达与缺氧的关系；在细胞缺氧、HAPH动物模型制备基础上，通过Real-time PCR检测细胞缺氧模型中apelin mRNA和miR-322变化，Western blot检测apelin蛋白表达变化、MTT实验、平板划痕实验检测miR-322的抑制对体外培养细胞的活性的影响；通过HE染色检测HAPH动物模型肺组织变化，Real-time PCR检测肺组织中miR-322和apelin mRNA基因表达，免疫荧光观察组织中apelin、CD31和α-SMA表达变化。免疫组化检测apelin表达情况，Western blot 检测大鼠肺组织中apelin蛋白的表达。在对高原缺氧大鼠肺水肿组织测序数据深度分析后，结果发现TCONS_00059579、miR-322、Apelin之间存在着关联性，miR-322初步抑制实验证实miR-322降低后，TCONS_00059579、Apelin表达水平升高，三种RNA水平的变化趋势，表明TCONS_00059579可能通过竞争性抑制miR-322实现对Apelin的调控；细胞缺氧模型和HAPH模型结果显示，apelin和miR-322在低氧条件下表达共同上调，miR-322抑制了低氧状态下体外细胞中apelin的表达，miR-322的抑制改善了体外培养细胞的活性。成功构建大鼠PAH模型，miR-322的抑制缓解了大鼠PAH的症状。本研究为HAPH的防治提供新靶点与新策略。