富集于肺癌细胞外囊泡中的YRNA片段的选择性分拣/分泌机制及其功能研究

YRNA is a class of non-coding small RNA particularly plentiful in extracellular vesicles (EVs)-derived various cell type, it is poorly understood for EVs biogenesis, cargo selective sorting and functional mechanism of YRNA. Our previous studies show that, YRNA fragments hY4F and hY4rF are highly enriched in EVs-derived lung cancer patients’ peripheral blood; RNA pulldown and western blot results show that, in lung cancer cells A549, YBX1, a kind of RNA binding protein, can specifically bind to the YRNA fragments hY4F and hY4rF, and YBX1 expression downregulation by siRNA cause the accumulation of YRNA fragments hY4F and hY4rF in A549 cells, thus for YRNA fragment been selective sorting / secrete into extracellular vesicles YBX1 is required. In addition, the mRNA and protein expression of Rab27b, an EVs secreted-associated protein, is unexpectedly high in lung cancer cells than IMR-90 cells. Rab27b expression downregulation by siRNA affects cellular proliferation in A549 cells. In this project, we will further clarify the molecular mechanism for YBX1 regulating the selective sorting of YRNA fragments hY4F and hY4rF into lung cancer cells EVs, and for Rab27b/Rab27 effector protein regulating EVs secretion. And we also explore how YBX1/Rab27b regulating the selective sorting / secretion of YRNA fragments hY4F and hY4rF into EVs affect the biological function of the EVs-donor cells and EVs-receptor cells, and its molecular mechanism, and further verified these regulatory functional roles in animal level by nude mice. In our knowledge, this project will at first reveal the molecular mechanism and functional roles of YBX1 regulating the selective sorting / secretion of YRNA fragments into EVs.

YRNA是一类能高丰度富集于细胞外囊泡（EVs）的非编码小RNA，但在其EVs形成、货物分拣机制和功能方面，还知之甚少。我们的前期研究表明：YRNA片段hY4F和hY4rF高丰度富集于肺癌EVs中；肺癌细胞A549中一种RNA结合蛋白YBX1能特异性结合YRNA片段，其表达下调造成YRNA片段在A549中的积累，因此YBX1对YRNA片段被选择性泌出胞外是必须的；一种与EVs分泌相关基因Rab27b在肺癌细胞中异常高表达，其表达下调影响肺癌细胞增殖。本项目将阐明YBX1调节YRNA片段选择性分拣到EVs中的分子机制；明确Rab27b/Rab27效应器蛋白调节EVs分泌的分子机制；探讨YBX1/Rab27b调节YRNA片段选择性分拣/分泌产生EVs对供体细胞和受体细胞的功能影响和机制，并从动物水平进行功能验证。本项目将首次揭示YBX1通过特异性结合YRNA调控其选择性分拣/分泌的机制和功能。

YRNA是一类能高丰度富集于细胞外囊泡（EVs）的非编码小RNA，但在EVs形成、货物分拣机制和功能方面，还知之甚少。本项目的前期研究表明：YRNA片段hY4F和hY4rF高丰度富集于肺癌EVs；肺癌细胞A549中一种RNA结合蛋白YBX1能特异性结合YRNA片段，其表达下调造成YRNA片段在A549中的积累，因此YBX1对于YRNA片段被选择性泌出胞外是必须的；一种与EVs分泌相关基因Rab27b在肺癌细胞中异常高表达，其表达下调影响肺癌细胞增殖。在本项目的运行过程中，我们进一步阐释了YBX1调节YRNA片段选择性分拣到EVs中的分子机制；明确了Rab27b/Rab27效应器蛋白调节EVs分泌的分子机制；探讨了YBX1/Rab27b调节YRNA片段选择性分拣/分泌产生EVs对供体细胞和受体细胞的功能影响和机制，并从动物水平进行了功能验证。进一步地，我们还在本项目研究基础上进行了扩展研究。一方面针对肺癌合并病毒感染过程进行了初步研究，结果显示，hY4F能够促进病毒感染诱导的肺癌细胞中炎症因子的表达、病毒感染影响肺癌细胞外囊泡hY4F和IL-6 mRNA的分泌、而YBX1能够调控hY4F对病毒感染的响应；另一方面我们也针对缺氧条件下肺癌细胞外囊泡中的内容物的变化及其功能进行了初步探讨。本项目首次揭示了YBX1通过特异性结合YRNA调控其选择性分拣/分泌的机制和功能，同时也对本项目的后期研究工作提供了重要的实验依据。