Exosome介导小RNA进入细菌并跨界调控基因表达的机制研究

Small RNAs are non-coding regulatory RNAs that can silence genes with complementary sequences in a mechanism called RNA interference (RNAi). The RNAi machinery is conserved in most eukaryotes and mediated by non-coding small interfering RNAs (siRNAs), microRNAs (miRNAs) and piwi-associated RNAs (piRNAs). RNAi functions not only as a defense mechanism to silence foreign DNA and RNA species such as those from viruses, transposons, and transgenes, but also plays an important role in regulating and fine-tuning the expression of genes in a plethora of diverse physiological and cellular processes. Mobile small RNAs that translocate within an organism have been observed in various plant and animal systems. Some small RNAs can even move across the boundaries between hosts and their interacting pathogenic, parasitic, or symbiotic organisms and trigger gene silencing in the unrelated species from different kingdoms, a mechanism termed cross-kingdom RNAi. Indeed, interaction with other organisms by way of cross-kingdom RNAi has been observed in plant and animal systems. However, small RNAs induced cross-kingdom regulation between mammals and bacteria has been rarely reported in recent years. In mammals, functional small RNAs circulate through body fluids, often encapsulated in vesicles called exosomes, and exosomes have been reported to function as the vehicles, delivering various effectors or signal molecules between specific cells and tissues. In our preliminary work, we found that exosome could deliver molecules, including small RNAs, to bacteria. Based on the compelling result, we aim to explore whether host small RNAs within mammal cells-derived exosomes could be delivered into bacteria, to function in gene regulation. Furthermore, we will investigate the mechanisms controlling exosome-mediated entry of small RNAs into bacteria, as well as the mechanisms by which small RNAs are processed after they enter bacteria and regulate target gene expression. This study is very important and intriguing, since the phenomenon of RNAi has not been reported in bacteria so far, and bacteria, such as E. coli, lack essential RNAi component, such as AGO and Dicer homologous proteins. Moreover, we will study the biological function of host small RNAs affecting the growth of gut bacteria, and the potential applications for the treatment of antibiotic-resistant bacteria. In conclusion, this project will increase our knowledge of small RNAs induced cross-kingdom regulation, demonstrate new roles for non-coding small RNAs in bacteria, and provide a particular perspective on the study of communications between mammals and bacteria.

非编码小RNA可以调节细胞内源基因表达，同时也是细胞间通讯的重要信号分子；进一步的研究表明，小RNA还参与了包括动物、植物和原生动物等物种间的跨界调控。在哺乳动物体内，小RNA借助运输载体exosome的包裹保护得以稳定地存在于循环系统中，并在细胞及组织间发挥信号传递的作用。在前期研究中，我们发现exosome也可作为运输载体，向细菌中运送包括小RNA在内的多种物质。在此基础上，本项目立意于系统研究由哺乳类细胞产生的exosome如何将胞内小RNA运输到细菌中，并发挥基因调控功能，从而影响细菌的生物学特性。进一步地，本项目将在分子水平上阐明小RNA在细菌胞内调控靶蛋白表达水平的机制。此外，我们还将探索小RNA影响肠道细菌生长的生物学功能，以及在抵抗耐药菌方面的潜在应用。本项目不仅有助于拓展我们对非编码小RNA跨界调控现象与机制的认识，还有望在理解人体与细菌之间相互作用关系提供新的切入点。

非编码小RNA可以调节细胞内源基因表达，同时也是细胞间通讯的重要信号分子；此外，小RNA还参与了包括动物、植物和原生动物等物种间的跨界调控。在哺乳动物体内，小RNA借助运输载体exosome的包裹保护得以稳定地存在于循环系统中，并在细胞及组织间发挥信号传递的作用。我们前期研究发现exosome也可作为运输载体，向细菌中运送包括小RNA在内的多种物质。本项目系统研究由哺乳类细胞产生的exosome如何将胞内小RNA运输到细菌中，并发挥基因调控功能，从而影响细菌的生物学特性。进一步地，本项目在分子水平上研究小RNA在细菌胞内调控靶蛋白表达水平的机制。此外，我们还研究小RNA影响肠道细菌生长的生物学功能，以及在抵抗耐药菌方面的潜在应用。我们发现，exosome是小RNA在哺乳动物细胞和细菌之间传递的有效媒介；通过exosome介导的小RNA进入细菌体内，能够调控细菌靶基因的表达，此基因沉默功能需要外泌体导入的AGO2蛋白参与，并以翻译抑制的方式执行；运用外泌体包裹靶向耐药基因的siRNA，我们实现了对超级细菌MRSA耐药性的调节，实施较低浓度抗生素即可抑制细菌生长，并在菌血症模型小鼠中成功应用该策略，保护小鼠免于MRSA感染引起的死亡。本项目不仅有助于拓展我们对非编码小RNA跨界调控现象与机制的认识，还有望在理解人体与细菌之间相互作用关系提供新的切入点，既有重要的生物学意义，也具有抗菌治疗的应用价值。