miR-362-5p靶向CYLD基因促进人NK细胞发育

Natural killer (NK) cells play key roles during early host defense against invading pathogens and tumors. However, little is known about the molecular basis of the development of human NK cells. In our preliminary data, we identified a novel microRNA that has never been reported to--miR-362-5p, which was overexpressed in mature human peripheral blood NK cells but down regulated in immature decidual NK cells. In addition,we identified that CYLD, a negative regulator of WNT/beta-catenin signaling, is a potential target of miR-362-5p. Because published data have shown that WNT/beta-catenin signaling supports human NK cell development, we hypothesized that miR-362-5p promotes human NK cell development by down-regulating CYLD. To test this hypothesis, we will conduct a series of experiments as follow: based on the in vitro human NK cell development experiment system, we used the lentivirus vector systems to investigate the regulatory roles of miR-362-5p and CYLD in human NK cell development.Moreover,we used FACS,qRT-PCR and Western blot to investigate the roles of miR-362-5p and CYLD in human NK cell function as well as the possible intracellular signaling mechanisms which were related to NK cell development and function. The success of this project will provide a new mechanistic explanation for regulation of NK cell development by miRNAs and ultimately implicate a potential application of miR-362-5p or CYLD in NK cell adoptive immunotherapy.

NK细胞具有抗肿瘤、抗感染等重要作用。但NK细胞发育的调控机制不清。前期通过microarray技术发现一个全新的miRNA-miR-362-5p特异性高表达于发育成熟的pNK细胞中，而在发育早期的dNK细胞中表达明显降低，进一步发现CYLD为其潜在靶基因。CYLD可抑制WNT/beta-catenin等信号的激活,而WNT/beta- catenin信号的激活可促进NK细胞发育。因此推测miR-362-5p靶向CYLD基因促进NK细胞的发育。为验证这一假设，本课题将构建miR-362-5p及CYLD上调和下调慢病毒载体，感染脐血CD34+细胞，结合体外NK细胞发育体系，观察miR-362-5p/CYLD对NK细胞发育、表型及功能的影响，qRT-PCR、Western blot检测NK发育相关信号的活化情况，阐明miR-362-5p促进NK细胞发育及相关分子机制，最终为NK细胞的生物治疗提供新思路。

微小RNA是一类在转录后水平调节基因表达的重要分子。然而，单个microRNA在自然杀伤（NK）细胞发育过程中的作用仍然知之甚少。本项目中，我们发现miR-362-5p表达水平对来自CD34 +造血干细胞的CD56 + NK细胞的发育及其功能有着重要的调控作用。我们进一步证明CYLD，NF-kB信号的负调节子，是NK细胞中miR-362-5p的直接靶基因。此外，miR-362-5p的过表达可以促进人原代NK细胞中IFN-γ，穿孔素，粒酶B和CD107a的表达，NK细胞效应功能增强。结合小干扰RNA沉默CYLD 可以模拟miR-362-5p在NK细胞中过表达的效应；抑制miR-362-5p在NK细胞中的表达则具有相反的效应，且CYLD siRNA可以中和其效用。以上结果表明miR-362-5p至少部分通过调控靶基因CYLD促进NK细胞的发育和功能。总之，本项目研究结果揭示了miR-362-5p在人类NK细胞生物学中起重要作用，miR-362-5p可能作为调节NK细胞发育和效应功能的重要靶点。