Gax基因靶向调节低氧性肺动脉高压的机制

In previous studies, we found that endogenous expression of Gax gene (growth arrest-specific homeobox) in pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC), which involved in hypoxia-induced pulmonary hypertension (HPH), was detected. However, over-expression of Gax gene via gene transfer had prevented the development of HPH and had anti-proliferative and pro-apoptotic effects on hypoxia-treated PASMC. However, the effects of over-expression of Gax on hypoxia-treated PAEC is still unclear. The project will build three models: animals, PAEC transfected with Gax gene, Gax gene transfection PAEC co-cultured with PASMC. All experiments will be completed under normoxic or hypoxic conditions. The characteristics indicators of HPH in the rats models will be detected. The cell proliferation and the levels of mRNA, protein, and DNA methylation of Gax, HIF-1α, ET-1, VEGF, eNOS, Notch1 and Jagged1 in the PAEC transfected with Gax gene will be evaluated, respectively. The PASMC proliferation and the levels of mRNA, protein, and DNA methylation of Gax, Notch1 and Jagged1 in the PAEC in the co-cultured model will be assessed, respectively. The regulation of RNA-binding protein HuR and ubiquitination on Gax and HIF-1α will be studied with PULL DOWN and IP methods. Variation of Gax and HIF-1α at the levels of alternative pre-mRNA splicing, epigenetic modifications, transcription, post-transcription, translation, post-translation will be investigated. The present study is to clarify the effects of Gax on PAEC and the novel regulation mechanisms of NF-κB/Notch/HIF-1 signaling pathways and their crosstalk. Thus, our project will provide a scientific basis for HPH mechanisms and targeted prevention and treatment strategy.

我们前期发现Gax基因在低氧性肺动脉高压（HPH）病理生理的肺动脉平滑肌细胞（PASMC）和内皮细胞（PAEC）中内源性表达，增强Gax基因表达防止HPH发生、调控PASMC增殖等，而其对PAEC的功能不明。本项目建三个模型：动物、Gax基因转染PAEC、Gax基因转染PAEC后与PASMC共培养。低氧处理前后检测大鼠HPH特征指标；单细胞模型PAEC增殖等及Gax、HIF-1α等mRNA、蛋白和甲基化水平；共培养PAEC内Gax、Notch1等mRNA、蛋白和甲基化水平，PASMC增殖等。用PULL DOWN和IP技术研究HuR、泛素化对Gax和HIF-1α调控，Gax和HIF-1α在可变剪接→表观遗传→转录→转录后→翻译→翻译后水平的变化规律与调控。明确Gax对PAEC的作用及NF-κB/Notch/HIF-1信号通路与串话调控新机制。为阐明HPH机制及研发靶向治疗新策略提供依据。

首先，本项目研究Gax基因转染对低氧性肺动脉内皮细胞（PAEC）增殖、凋亡和周期的影响。结果发现，低氧早期内皮细胞异常增殖加速，而此时增强Gax基因表达可抑制细胞异常增殖，抑制细胞从G0/G1期进入S期和G2/M期，诱导细胞凋亡；随着低氧时间的不断延长，细胞增殖受到抑制，而此时增强Gax基因表达却又激活细胞增殖，促进细胞从G0/G1期进入S期和G2/M期，抑制凋亡。Gax基因对维持内皮细胞数量的稳态具有双向调节作用。其次，观察Gax基因对低氧性PAEC中ET-1、VEGF和eNOS基因表达的影响。结果表明，低氧上调PAEC中VEGF和ET-1的基因表达，下调eNOS基因表达，而增强Gax表达可以逆转这种现象。再次，观察Gax基因对低氧性PAEC中HIF-1α基因表达的影响，结果显示，增强Gax基因的表达能下调低氧诱导的HIF-1α mRNA和蛋白表达，这与Gax基因调节低氧性内皮细胞异常增殖、凋亡和周期的机制相关。最后，观察Gax基因对低氧性PAEC中HIF-1α、ET-1、VEGF和eNOS DNA甲基化的影响，结果发现，Gax基因通过DNA甲基化途径调控上述基因的表达，这可能是Gax基因调节低氧性肺动脉高压的新机制。在SCI收录期刊、中文核心期刊和中国科技论文统计源期刊上发表论文7篇，并都标注有“国家自然科学基金资助”。本项目首次研究Gax基因调节低氧性PAEC生物学功能与机制，且取得一定的创新性成果。