猪高原低氧获得性习服相关的DNA甲基化组变异及其跨代遗传的表观调控

Over the centuries, the molecular mechanism of the adaption to high-altitude hypoxia in mammals has been a hot research topic for medical scientists, biologists and sociologists. It is of important implications for the prevention and treatment of mountain sickness, the development of animal husbandry in plateau and the protection of animal genetic resources. To date, relevant researches have been focusing on the long-term adaption of high-altitude hypoxia, and most of them based on the DNA sequence level. However, in recent years ceaseless reports demonstrated that, epigenetic mechanism (especially variation in DNA methylation level) was also an important factor contributing to the adaption (especially the fast acclimatization) to hypoxia in mammals. To solve this frontier and popular scientific problem, we proposed to employ low-altitude domestic pig as study material, construct and compare their single-base resolution genomic DNA methylation map of distinct tissues between pre- and post-acclimatization status, analyze that together with their gene expression profiles, and comprehensively elucidate the DNA methylation variation underlying the phenotypic variation during high-altitude hypoxia acclimatization. Simultaneously, in order to study the hereditability of the DNA methylation pattern that related to the adaption of high-altitude hypoxia, we proposed to trace the DNA methylation variation between two generations. Our results will improve and supplement the molecular regulation network of the fast acclimatization and genetic adaption of mammals to high-altitude hypoxia. We envision that these discoveries will provide new insights into the prevention and treatment of mountain sickness, and will provide scientific guidance for the pig breeding of high-altitude resistance.

高原低氧适应分子遗传机制的阐明对高原疾病的预防治疗、高原畜牧业的发展、动物资源保护等都具有重要意义。目前，哺乳动物高原低氧适应性的研究主要针对动物对低氧的长期“遗传适应”，且主要立足于DNA序列水平。然而近期研究表明，DNA甲基化水平的变异也参与了动物的低氧适应调节，尤其在动物对低氧环境的短期“获得性习服”方面。本课题针对动物的适应性进化这一热点科学问题，以移居高原前后平原家猪为研究对象，旨在构建高原低氧习服前后家猪各组织的单碱基分辨率全基因组DNA甲基化图谱，与基因表达谱进行关联分析，进而全面解析猪高原低氧“获得性习服”相关的DNA甲基化变异，并通过跨代跟踪研究探索性分析低氧适应相关DNA甲基化模式的可遗传性。其结果将从表观遗传学的角度，完善和补充哺乳动物高原低氧适应的分子机制，为人类高山疾病的预防与治疗提供新思路和视野，同时为猪以及其他农业动物的抗高原低氧育种提供科学指导。

长期以来，对哺乳动物高原低氧适应性的研究主要针对动物对低氧的长期“遗传适应”，且主要基于DNA序列水平。近期研究表明，DNA甲基化水平的变异参与动物的低氧适应调节，尤其在动物对低氧环境的短期“获得性习服”方面发挥重要作用。本课题针对动物的适应性进化这一热点科学问题，以荣昌猪为研究对象，建立“动物移居模型”，发现荣昌猪在高海拔妊娠环境下，初生仔猪线粒体DNA拷贝数在6个组织 (脑、肝、肺、肾、背最长肌和腰大肌) 中显著降低，在高低海拔猪的脑、心和肺组织中分别筛选出288个脑组高可靠性的差异lncRNA、15个心组织中高可靠性的差异lncRNA和9个肺组织中高可靠性的差异lncRNA；采集原生状态荣昌猪 (n = 3) 和移居平原荣昌猪 (n = 3) 的背最长肌组织，采用BS-seq技术构建荣昌猪骨骼肌的单碱基分辨率DNA甲基化图谱，并比较不同环境下骨骼肌可能存在的能量代谢差异；通过链特异性反转录和PCR发现了存在于猪基因组中PLA2G16基因座上的反义lncRNA (即PLA2G16 AS)，并活体组织表达相关性和离体细胞功能学分析，验证了PLA2G16 AS对PLA2G16基因的调控作用；以大鼠心肌细胞为模型，对外泌体测序分析结果发现，低氧显著改变了外泌体中miRNA种类，并从外泌体中鉴定出62个差异表达的miRNA，这些差异miRNA功能富集于HIF-1信号通路和凋亡相关信号通路；以心肌梗塞大鼠的心肌细胞作为模型，验证了miR-532-5p通过靶向作用于PDCD4抑制了低氧诱导的心肌细胞凋亡；以仓鼠前体细胞低氧处理模型，系统阐释脂肪组织低氧微环境对肥胖的进展。本项目在多种哺乳动物中研究低氧对脂肪、肌肉等组织的mRNA、miRNA和lncRNA的调控作用，在一定程度上解析了DNA甲基化、lncRNA以及microRNA对猪各组织高海拔适应性的的调控作用，为猪以及其他农业哺乳动物的抗高原低氧育种提供科学指导，也从表观遗传学角度，为人类高山疾病的预防与治疗提供可参考的基础数据。