小肠上皮细胞microRNA介导水苏糖调控肠道菌群的分子机制研究

A recent research demonstrates that the mice shape the gut microbiota via small intestinal epithelial cells-secreted fecal miRNAs. By performing 16S rDNA and Small RNA sequencing, we previously found that stachyose, a prebiotic food product, remarkably changed the gut microbial composition, increased the abundance of beneficial bacteria and suppressed that of destructive bacteria, and reprogrammed the miRNA expression profile of small intestine in mice. Thus, to clarify the small intestine miRNA-dependent novel prebiotic mechanism has become the primary bottleneck for scientifically cognizing the intestine-improving effect of stachyose. In this project, we propose to verify the predicted stachyose-induced abundance-altered gut bacteria and differentially expressed miRNAs using qPCR. At the same time, we will evaluate the impact of blocking the production of intestinal epithelial cell miRNA with RNA interference on the regulatory efficacy of stachyose on distal colonic microbiota. Moreover, we will explore the roles and modes of action of intestinal epithelial cell miRNAs on the growth of stachyose-induced abundance-altered gut bacteria by establishing miRNA and bacteria-based partial least-squares regression model and conducting co-culture experiments of miRNA-bacteria. Overall, this study aims to illustrate the intestinal epithelial cell miRNA-mediated molecular mechanisms underlying the regulative effect of stachyose on gut microbiota, and uncover novel target miRNAs for the prebiotic function of stachyose. This study will provide scientific bases for perfecting the prebiotic nutritional theory and for innovating prebiotics-based novel foods that control gut homeostasis by targeting miRNAs.

新近证实，小鼠能够通过小肠上皮细胞分泌的miRNA特异性调控肠菌群结构。申请人前期利用16S rDNA和小RNA测序亦发现，益生元食品水苏糖显著改变小鼠肠菌群组成，增殖有益菌抑制有害菌，并重编小肠miRNA表达谱。明晰水苏糖的小肠miRNA依赖性益生新机制已成为科学认知其益肠功能首要解决的瓶颈问题。本项目拟采用qPCR技术探析水苏糖诱导的丰度改变细菌和差异表达miRNA；利用RNA干扰技术考察上消化道小肠上皮细胞miRNA合成受阻对水苏糖调节远端结肠菌群效力的影响；通过构建基于miRNA与细菌的偏最小二乘回归效应关系模型并结合miRNA-细菌共培养实验，探究miRNA对各丰度改变细菌的生长调控作用及机理。阐明小肠上皮细胞miRNA介导水苏糖调控肠菌群的分子机制，揭示水苏糖肠益生功能的miRNA新靶标，为完善益生元营养理论并为创新基于益生元靶向miRNA调控内环境稳态的新型食品提供科学依据。

水苏糖被长期认为因不被机体消化酶水解而穿越上消化道，直达肠道后端发挥传统益生功效。然而，我们首次揭示水苏糖除重塑肠道菌群结构外，亦重编小肠细胞分泌至粪便的miRNA表达谱。为明确这些miRNA是否参与水苏糖对肠道微生物的调控，本研究联合16S rDNA和Small RNA高通量测序技术，系统考察了水苏糖对肠道菌群组成和小肠miRNA表达谱的影响，筛选了水苏糖诱导的丰度显著改变的肠道细菌和表达量显著改变的miRNA，通过构建PLSR偏最小二乘回归定量效应预测模型对差异miRNA和差异细菌进行相关性预测，最后利用流式细胞术和微生物体外发酵技术进行结果验证。.首先，水苏糖诱使小鼠肠内微生物多样性、物种分布及聚类方式发生明显改变，其中Lactobacillus gasseri、Lactobacillus alimentarius和Akkermansia muciniphila等8种菌的丰度发生显著变化。同时，水苏糖显著改变小鼠小肠组织中27种miRNA的表达水平。进一步以这些表达显著改变的小肠miRNA为自变量，以丰度显著改变的肠道菌属为因变量，成功构建PLSR偏最小二乘回归模型，结果预示这三种菌的相对丰度与多种差异miRNA水平呈现显著相关关系。综合小肠组织和粪便中各关联性miRNA的表达量，初步预测被水苏糖显著下调的miR-133b-3p和miR-3068-5p可能与这三种有益菌显著相关。最后，将化学合成的miRNA模拟物与细菌共培养后发现，miR-133b-3p和miR-3068-5p均可进入这三种细菌菌体内，其中miR-133b-3p可显著抑制Lactobacillus gasseri的体外增殖。令人惊奇的是，我们还拓展发现水苏糖积聚小肠上皮细胞膜改变其外泌体miRNA组成的营养新功能。.据此，我们推断：水苏糖可能通过下调小肠细胞分泌的miR-133b-3p水平特异性促进肠道有益菌Lactobacillus gasseri定植，进而改善小鼠肠道微生物组结构。当前已完成项目合同拟定的全部任务，上述取得的相关研究成果在农业食品领域J. Agric. Food Chem.、Food Funct.等国内外高水平期刊发表研究论文7篇，且受到国内外学术界高度关注。此外，部分研究成果正在整理，拟发表在更高水平的期刊。