线粒体基因组有害突变在藏族低氧适应中的作用及机制研究

Tibetans, dwelled in Tibetan Plateau, are considered highly adaptive to severe hypoxic environments. Genome sequencing analysis found that mutations in EPAS1 and EGLN1 genes play an important role in hypoxia adaptation in Tibetans. Our previous work found that: 1) potentially pathogenic and deleterious mitochondrial DNA mutations are enriched in Tibetan highlanders; 2) In addition, in our study on four Tibetan populations, we observed that the proportion of pathogenic mutations increased with the elevation of altitudes and the mutation frequencies of four SNPs located in EPAS1 were significantly associated with altitudes. Both of mitochondrial DNA deleterious mutations and nuclear gene mutations play an important role in Tibetans, but the interaction between them is not yet clear. In this study, individual samples of wild-type/mutant of EPAS1 and EGLN1, as well as wild-type/mutant of mitochondrial DNA, were collected from different altitudes to construct cybrids, immortalized cell lines and pluripotent stem cell lines. Mitochondrial functions will be tested in different concentration of oxygen to clarify the interaction and mechanism between nuclear genes EPAS1/EGLN1 and mitochondrial DNA and figure out the potential mechanisms. The accomplishment of this work would help to reveal the mechanism of hypoxia adaptation in Tibetans.

藏族人群已经良好地适应了高原的低氧环境。全基因组测序分析发现EPAS1和EGLN1两个基因上的突变对藏族人群的低氧适应有重要作用。我们的前期工作发现：1）在包括藏族的高原世居民族线粒体DNA中富集了平原人群中致病的突变；2）在四个不同海拔水平的藏族群体中，线粒体DNA所产生的有害突变的比例随着海拔的上升而增加，并且核基因EPAS1的4个SNP位点的突变频率与海拔水平显著相关。线粒体DNA有害突变和核基因突变在藏族的低氧适应中均起了重要的作用，但是两者的互作关系尚不明确。本研究拟在不同海拔的群体中采集EPAS1与EGLN1野生型/突变型，以及线粒体DNA野生型/突变型的个体样本，构建融合细胞系、永生化细胞系及多潜能干细胞系，在不同氧气浓度条件下进行线粒体功能实验，阐明核基因EPAS1/EGLN1与线粒体DNA的互作及机制。本项目的完成将有助于揭示藏族人群高原低氧适应的机制。

西藏高原是含氧量低的极端环境，平原世居者会产生一系列低氧诱导反应，而青藏高原的世居族群已经适应了高原的低氧环境。前期研究发现EGLN1和EPAS1在藏族与汉族人群中有显著差异，且藏族群体的线粒体DNA单倍型多为M9a单倍型。核基因和线粒体基因的互作可能帮助藏族更好地适应高原低氧环境。为了进一步探讨藏族如何适应高原低氧环境，我们通过构建平原和高原融合细胞模型，并在其基础上进一步通过慢病毒感染的方法改变其核基因背景，然后在常氧（21%O2）以及低氧（1%O2）条件下检测细胞增殖、线粒体功能、线粒体复合体与超级复合体的表达水平以及线粒体逆向信号通路等探讨藏族低氧适应的具体机制。我们发现与平原对照M7/8及高原M9a单倍型相比，M9a单倍型继续构建的EPAS1下调细胞系的线粒体功能最好、细胞增殖能力最高且凋亡水平最低。机制上，M9a+shEPAS1会增强线粒体耗氧量，进一步“moderate”促进ROS的生成，从而稳定HIF-1A的表达，进一步促进BNIP3/NIX介导的线粒体自噬作用，且会减少整体的炎症水平。且ROS会作为“Signal”来抑制DUSP1的表达，促进PAK3的表达来激活AMPK、MAPK等信号通路。进一步证明了藏族适应性单倍型M9a加上适应性核基因EPAS1下调具有“强强联合”的作用来适应高原低氧环境。本项目首次发现藏族适应性线粒体以及核基因在增强线粒体功能的同时会促进ROS的生成，ROS可以作为信号激活HIF-1A-BNIP3/NIX介导的线粒体自噬以及相关AMPK及MAPK等逆向信号通路，具有更好的线粒体功能以及细胞增殖能力以更好地适应高原低氧环境。本项目的实施为高原藏族如何适应高原低氧环境提供了新的机制和解释。并推动我们对其他低氧相关疾病机制的理解，对揭示藏族低氧适应的机制及低氧相关疾病的理解具有重要意义。