调控肿瘤逃逸NK细胞杀伤的miRNA的发现及机制研究

The mechanisms that have been developed by tumor cells to evade NK cell-mediated lysis are not well understood.We are still at the very beginning of the study on what miRNAs regulate the expression of ligands on the surface of tumor cells to modulate tumor susceptibility to NK cells. To address this question, we proposed to perform microRNA deep sequencing and high-throughput whole-genome sequencing on a pair of prostate cancer cell lines that have different susceptibilities to NK cells. According to the sequecing results followed by the verification with biological function assays, we have found that overexpression of miR-296-3p in tumor cells significantly suppressed NK cell cytotoxicity and intercellular adhesion molecule-1 (ICAM-1) which has been identified by us as a new target for miR-296-3p may be involved in this process. Based on these findings, we will examine the effects of miR-296-3p on immune evasion, tumorigenesis and tumor metastasis at molecular, cellular, animal and clinical levels and elucidate the mechanisms involved. miR-296-3p will also be evaluated as a target for anti-tumor therapeutics. A successful outcome will advance our understanding of the mechanisms for immune evasion and provide new target and insight into anti-tumor immune therapy based on NK cells.

关于肿瘤细胞如何逃逸NK细胞杀伤的具体分子机制目前尚未完全清楚。哪些miRNA调控肿瘤细胞表面的配体表达从而介导肿瘤细胞逃逸NK杀伤，国内外研究刚刚起步。本项目拟通过对NK细胞杀伤具有不同敏感性的配对前列腺癌细胞系的miRNA深度测序和基因表达谱高通量测序，找到调控肿瘤细胞逃逸NK杀伤的关键miRNA和蛋白。通过测序和初步的生物功能学验证，我们发现miR-296-3p在肿瘤细胞中的过表达能够抑制NK细胞对其杀伤,可能通过我们新鉴定的靶蛋白ICAM-1实现。在此基础上，我们将从分子、细胞、动物和临床标本这四个层面，系统地检测miR-296-3p与肿瘤细胞逃逸NK杀伤、肿瘤发生和肿瘤转移之间的关系，并考察ICAM-1是否为miR-296-3p发挥上述功能的关键效应分子,从而找到调控肿瘤细胞逃避NK细胞杀伤的新分子机制。最后，我们也将初步探索miR-296-3p成为治疗靶点的可能性。

关于肿瘤细胞如何逃逸NK 细胞杀伤的具体分子机制目前尚未完全清楚。哪些miRNA 调控肿瘤细胞表面的配体表达从而介导肿瘤细胞逃逸NK 杀伤，国内外研究刚刚起步。在本项目的研究中，我们首先观察到人体外扩增的NK细胞对前列腺上皮细胞系P69和其衍生的恶性转移瘤细胞系M12具有差异性免疫应答，通过对P69和M12表达的NK细胞激活和抑制性配体分析，发现在P69中高表达而M12中低表达的ICAM-1可能介导了该现象。当在P69中过表达miR-296-3p时会导致ICAM-1下调并引起对NK细胞的抵抗，而在M12沉默miR-296-3p后则引起ICAM-1上调和对NK细胞的敏感。随后通过荧光素酶报告基因实验证实miR-296-3p可以结合到ICAM-1的3’UTR并抑制其表达。当进一步在过表达miR-296-3p的P69中回复ICAM-1的表达时可以逆转其对NK细胞的抵抗，而利用shRNA技术在沉默miR-296-3p的M12中敲低ICAM-1则可以回复对NK细胞的抵抗。同时人前列腺肿瘤组织中miR-296-3p的相对高表达和它与ICAM-1的显著负相关表达也为miR-296-3P可能是致癌miRNA和miR-296-3p调控ICAM-1提供了佐证。更重要的是，当将表达荧光素酶（luciferase）或绿色荧光蛋白（GFP）的M12细胞系通过尾静脉注射到裸鼠并在特定的时间点分别进行动物活体成像或外周血流式检测后发现，肺部转移或外周血中沉默miR-296-3p的M12较对照组和敲低ICAM-1组更容易被清除，而预先用特异性抗体清除裸鼠内源性NK细胞后，各组间则不表现这种清除差异。随着再次经静脉注入人NK细胞后，上述差异再次出现。上述的体内和体外实验证实了miR-296-3p-ICAM-1这一新的信号轴通过介导外周血中肿瘤细胞的存活而影响了前列腺肿瘤的迁移，这为前列腺肿瘤的临床治疗和预后提供了新的靶点。