长链非编码RNA MEG3在缺氧诱导的肺血管重构中的作用

The proliferation of pulmonary artery smooth muscle cells (PASMCs) is the most significant feature of hypoxic pulmonary hypertension, however, the exactly mechanism for PASMCs proliferation remains unclear leads to the less therapy efficiency of current drugs for pulmonary hypertension. The roles of long non-coding RNA (lncRNA) in cardiovascular diseases have made a great progress, whereas lncRNA contributes hypoxic pulmonary hypertension and the underling mechanism still unknown. Our previous results have demonstrated that the expression of lncRNA maternally expressed gene 3 (MEG3) and pim-1 were increased, whereas microRNA-328 (miR-328) expression was decreased in pulmonary arterials from hypoxic pulmonary hypertension mice. According to the Target Scan software prediction, there is complementary base between lncRNA-MEG3 and miR-328, miR-328 and the mRNA of pim-1. These results lead us to hypothesis that “hypoxia increases the expression of lncRNA-MEG3, lncRNA-MEG3 targets to miR-328 and inhibits miR-328 expression, the down-regulation of miR-328 leads to the enhanced expression of pim-1, which contributes to PASMCs proliferation, finally leading to pulmonary vascular remodeling”. This study will employ microscale thermophoresis, liposome lung targeting drug delivery and other molecular biology technical to confirm our hypothesis. The results of this study will determine the role of lncRNA MEG3 in hypoxic PASMCs proliferation, and provide new clinical prevention and treatment schemes for pulmonary arterial hypertension.

肺动脉平滑肌细胞（PASMCs）增殖是缺氧肺动脉高压的最显著特征，其机制不清楚是导致临床上该疾病药物疗效不佳的最主要原因。长链非编码RNA（lncRNA）在心血管疾病研究中取得了重要进展，但lncRNA在缺氧肺动脉高压中的作用及机制仍不清楚。前期结果显示缺氧肺动脉高压小鼠肺动脉中lncRNA 母系表达基因3（MEG3）和pim-1表达升高，而miR-328表达下降；生物信息学软件预测出MEG3与miR-328，miR-328与pim-1具有结合可能性。因此本课题假设“缺氧诱导MEG3表达升高，MEG3负向调控miR-328，使miR-328调控pim-1表达的能力降低，从而引起PASMCs增殖、肺血管重构”。本课题拟采用微量热泳动技术、脂质体肺靶向给药等技术证实上述实验假设，确定lncRNA MEG3在缺氧PASMCs增殖中的作用，为临床防治肺动脉高压提供新思路。

肺动脉高压(Pulmonary Hypertension, PH)是一种以肺动脉压力明显且持续升高为主要特征的致死性疾病。目前认为PH的发病是因为肺动脉血管收缩以及肺动脉血管重构，导致肺动脉血管出现绝对和相对的狭窄，最终升高肺动脉压。PH的确切病因和发病机制尚不明确。本成果针对上述问题阐明了以下3点研究内容：①本研究明确了lncRNA MEG3缺氧动物模型和体外缺氧肺动脉高平滑肌细胞中的表达改变，及其lncRNA MEG3 缺氧条件下调控PASMCs 增殖中的作用，解决了我们提出的lncRNA参与缺氧肺动脉平滑肌细胞增殖的关键科学问题；②明确了lncRNA MEG3 与miR-328 的相互作用关系，明确microRNA是lncRNA的下游作用分子和关键节点；③明确了lncRNA MEG3 与miR-328 的相互作用对PASMCs 增殖的影响，揭示了lncRNA MEG3/miR-328 信号通路在缺氧肺动脉平滑肌细胞增殖、缺氧肺动脉高压中的作用。本成果显示lncRNA MEG3是缺氧肺动脉高压的关键调节因子，发现lncRNA MEG3在缺氧状态下上调，参与了缺氧诱导的肺动脉平滑肌细胞增殖和细胞周期进展。lncRNA MEG3的NT 2071–2094位点直接与miR-328-3p结合，下调miR-328-3p，导致IGF1R表达增加，并调节缺氧肺动脉高压的发生发展。这一发现表明lncRNA与缺氧肺动脉高压有关，并提示调节lncRNA MEG3的活性及其下游靶点miR-328-3p，是治疗这种致命疾病的一种新的治疗方法。另外，该成果还发现R8修饰脂质体的给药系统具有肺靶向传递的能力，使用R8修饰脂质体包裹lncRNA MEG3的siRNA，肺靶向干扰lncRNA MEG3表达。脂质体修饰siMEG3在1小时内肺部的生物分布达到了很高的水平，并保持24小时持续作用。因此，研究人员在缺氧治疗期间重复使用脂质体修饰siMEG3。实时定量PCR和FISH证实了靶向敲除效率。相反，非修饰siRNA和脂质体显示无明显效果，而且，右心室收缩压和右心指数间接证明脂质体修饰siMEG3能够降低肺动脉压。因此，使用脂质体修饰siMEG3敲除肺lncRMAMEG3表达有望成为新的治疗肺动脉高压的途径。该研究结果为肺动脉高压疾病发病机制的理解提供新视角，对肺动脉高压疾病防治提供了新途径。