猪体内食物源性植物microRNA对内源靶mRNA基因的调控

Are we controlled by what we eat? A recent publication by Zhang et al. in Cell Research at September, 2012 opened a world-wide discussion on the evolutionary impact of environmental dietary nucleic acid effects across broad phylogenies. This report described the uptake of plant-derived miRNA into the various internal organs in mammals following consumption of plant food, as well as apparent gene regulatory activity in vivo. Given the number of questions evoked by subsequent reports, the insufficient understandings of mechanism underlying the absorption and processing of exogenous plant-derived dietary miRNAs, we felt further evaluation of miRNA uptake and the potential for cross-kingdom gene regulation in animals was warranted to assess the prevalence, impact and robustness of the phenomenon. Herein, we aim to deciphering the biological significance of the uptake of plant-derived miRNAs through food and the molecular mechanism by which plant miRNAs are absorbed and processed in a pig model with improve experimental designs. We also try to test a possible mechanism of genetic exchange between the mother (i.e. pregnant sows) and fetus which conducted by exosome-mediated transfer of miRNAs. Our results will provide the evidences of the uptake of ingested plant miRNAs by pig, the regulation of target mRNA gene levels in various organs and plasma or phenotypic changes in pig from ingested plant miRNAs that would be indicative of target gene regulation after plant feeding. We envision that these discoveries will extend our acknowledgements of plant miRNAs and their roles in the biology of mammalian cells and animal organs. Equally important, the molecular understanding of cross-kingdom regulation of a porcine transcript in growth and development will be an important step forward for maximizing the economic benefits in producing high quality pork products for the benefit of human society.

2011年9月至今, miRNA在动植物间的"跨界"传递 --"食物来源的植物miRNA能够通过哺乳动物的消化器官进入血液循环系统, 进而跨物种地调控动物自身基因的表达", 已然成为一个存在较大争议的科学发现。本课题针对这一前沿热点科学命题, 提出一系列有趣而迷人的生物学假设, 进行全面深入的探索性研究。旨在鉴定猪体内食物源性植物miRNA的种类, 解析其可能的吸收方式、运输途径和富集规律, 明确其对内源靶mRNA基因的表达调控机制, 分析其对猪表型性状的影响类型和程度, 并探索性分析植物miRNA能否借助于外泌体实现在母猪和胎儿之间的传递。其结果将为进一步揭示物种间miRNA的传递机制和靶向基因表达调控提供新证据, 鉴定出的植物miRNA及其猪靶mRNA基因, 将为深刻理解猪经济性状的分子调控机制提供新思路和视野, 并有望在相关领域产生重要影响。

近年来，越来越多的研究发现某些植物miRNA具有抑制流感病毒、抑制乳腺癌的生长及参与调节蜜蜂发育等生物学功能。这些研究结果拓展了我们对动植物间的“跨界”交流的认知。但人们对食物来源的植物miRNA的吸收规律、分布特点及其潜在的生物学功能的认识还十分有限。猪作为重要的农业经济动物，其饲料含有高比例植物性成分，并且具有与人类相似的消化系统结构、代谢特征、心血管系统以及器官大小，使其成为理想的人类医学模型。本项目主要以猪为动物模型研究植物miRNA的吸收规律、组织分布特点及其潜在的生物学功能。首先采用高通量测序技术从玉米果实和大豆等11种可食用植物的微囊泡中分别鉴定124和445个植物miRNA，筛选出18个具有代表性玉米miRNA用于后续研究。对玉米进行高温高压处理，miRNA定量分析证实植物miRNA在体外的稳定性。给猪饲喂新鲜玉米后检测猪各种组织器官和体液中的玉米miRNA，本项目揭示了18种玉米miRNA在猪12种组织，5种玉米miRNA在猪胎儿的2种体液和7种组织中的分布规律。通过高碘酸氧化实验和TA克隆测序的方法进一步验证猪体内存在的植物miRNA。采用翻转肠囊实验方法在离体条件下研究肠道对植物miRNA的吸收特点，结合猪采食新鲜玉米和灌喂人工合成的植物miRNA后其血液中植物miRNA的定量结果，从离体和在体两个水平揭示植物miRNA的吸收规律。为进一步鉴定这些植物miRNA的潜在功能，我们针对植物中具有代表性、高丰度的miRNA以及猪体内食物源性植物miRNA，使用target Scan等软件预测其在动物体内的潜在靶基因，靶基因功能富集结果显示这些潜在靶基因主要涉及免疫、生长发育和疾病等过程。针对筛选出的具有潜在研究价值的miRNA及其靶基因，我们通过双荧光素酶报告基因系统和MirTrap免疫共沉淀系统明确了zma-miR164a-5p、miR-168c、miR-8155与其靶基因之间的靶标关系和作用位点。上述结果证实了食源植物miRNA能够通过消化道进入猪体内，并且这些进入体内的植物miRNA具有潜在的调节猪内源靶基因的功能，这些发现为进一步揭示植物miRNA跨物种的传递机制和靶向基因表达调控提供了新证据。