PAHRF/miR-23a/MST1构成ceRNA网络在肺动脉平滑肌细胞增殖和肺血管重构的作用机制

Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance and obliterative pulmonary vascular remodeling which mechanism is complicated and still unknown. Our studies discovered that lncRNA PAHRF (pulmonary arterial hypertension related factor) was low-expressed in PAH patients and pulmonary arteries in rats exposed to hypoxia. Bioinformatic analysis and experiment data indicate that there is miR-23a response element in PAHRF nucleic acid sequence. Further study found MST1 (mammalian Sterile 20-like kinase 1), was a novel target of miR-23a mediated the pulmonary artery smooth muscle cells proliferation. These results suggest that lncRNA PAHRF participates in pulmonary vascular remodeling by modulated miR-23a targeting of MST1 in PAH. We will utilize gene microarray, dual luciferase reporter gene assay and molecular biology approaches: i) to determine the clinical significance and function of lncRNA PAHRF in pulmonary vascular remodeling; ii) to reveal the miR-23a regulates mechanism of pulmonary artery smooth muscle cells proliferation through targeting MST1; iii) to identify the lncRNA PAHRF influences MST1 gene expression and function; iiii) to verify whether lncRNA PAHRF acts as a competing endogenous RNA by sponging miR-23a to regulate MST1 expression mediated pulmonary vascular remodeling. The aim of this study is probably going to provide a new explanation and give a further understanding of the occurrence of vascular remodeling in PAH from the perspective ceRNA hypothesis. This study may provide an effective therapeutics target to be considered in the clinical management of PAH.

肺动脉高压（PAH）是以血管增殖为主要特征的心血管疾病,病因未明。前期发现lncRNA PAHRF(PAH related factor)在PAH患者和缺氧肺血管低表达,PAHRF序列中存在miR-23a应答元件且miR-23a靶向调节MST1与平滑肌增殖有关。提示lncRNA PAHRF通过miR-23a介导MST1调控PAH血管重构。拟通过基因芯片、荧光素酶报告基因、分子生物学等，①明确lncRNA PAHRF在PAH肺血管重构的临床意义及功能；②揭示miR-23a通过靶MST1调控平滑肌增殖的机制；③确定lncRNA PAHRF影响MST1基因表达及功能；④验证lncRNA PAHRF是否作为竞争性内源性RNA(ceRNA)参与miR-23a调控MST1介导肺血管重构。本课题旨在从ceRNA假说角度为PAH血管重构的发生提供全新解释机制，为该致死性疾病的诊断和治疗提供新的治疗靶标。

肺动脉高压（PAH）是一种罕见的致命疾病，涉及由肺动脉平滑肌细胞（PASMCs）增殖增强介导的肺动脉血管重塑。越来越多新的研究表明lncRNA在PAH的发生发展中起重要作用。然而，一些新的lncRNA在人PAH中发生发展的机制仍缺乏研究。我们通过芯片分析并结合lncRNA数据库选择了在PAH中异常表达的lncRNA，发现lncRNA NONHSAT169231.1在PAH患者PAs中低表达，我们将这一lncRNA命名为肺动脉高压相关因子（pulmonary arterial hypertension related factor，PAHRF）。但是，PAHRF在PAH中的生物学功能仍然未知。接下来，我们运用realtime PCR验证PAHRF在体外表达。结果发现缺氧条件下PAHRF在PASMCs低表达，PAF和PAECs两种细胞缺氧没有显著变化。然后，我们通过荧光原位杂交分析了lncRNA PAHRF在PASMCs表达和定位，发现PAHRF位于人PASMCs的细胞质和细胞核中。通过MTT，CCK-8，EDU染色，JC-1测定，流式细胞术和蛋白质印迹检测PAHRF对PASMCs增殖和凋亡的影响。荧光素酶活性测定用于鉴定PAHRF/miR-23a-3p/丝氨酸/苏氨酸激酶4（STK4/MST1）相互作用。研究发现过表达PAHRF后抑制了PASMCs的增殖并促进其凋亡。基因敲除掉PAHRF后发挥相反的作用。萤光素酶活性测定证明了PAHRF和hsa-miR-23a-3p之间存在相互作用。另外,萤光素酶活性测定和生物信息学及分子生物学实验证实MST1是miR-23a-3p的靶基因。在此基础上，我们进一步探索了调控miR-23a-3p表达的分子机制，发现lncRNA PAHRF充当miR-23a-3p的内源海绵，沉默掉lncRNA PAHRF可以上调miR-23a-3p的表达。该研究揭示了由PAHRF，miR-23a-3p和MST1组成的PAH发病分子机制新型调节模式。本项目旨在从竞争性内源性RNA(ceRNA)假说角度为PAH患者血管重构的发展提供全新解释机制。该研究有助于理解lncRNA PAHRF的生物学特征，有助于理解lncRNA PAHRF在PAH血管重构中的作用及分子机制，并可能为PAH的防治提供新线索和潜在的干预靶标。