miR-128异常调控肿瘤浸润免疫细胞亚群促进胃癌免疫逃逸的机制研究

Tumor infiltrating lymphocyte (TILs) is one of the most crucial components of the tumor infiltrating immune-cell in tumor microenvironment. Previous studies showed that TILs can exert both tumor suppressive and promoting effects, activated CTLs correlate with better survival in some cancers , Treg cells promote tumor immune escape through suppression of anti-tumor immune responses. The mechanism through which TILs may enhance tumor progression is the production of cytokines that by tumor cells can affect TILs subgroups. However, the regulatory mechanisms of Tumor-TILs signaling are only partially known. In our preliminary studies, we found that aberrantly expressed miR-128 results in increased proportion of a T cell subpopulation that is CD4+FoxP3+. We bioinformatically identified three cytokine genes, IL16, SEMA4C and STC1,are downregulated in low miR-128 expressed GC in TCGA dataset. Mechanismly, miR-128 negtively regulated IL16 expression by directly bind to two motifs located in the 3’UTR region of IL16, and IL-16 stimulation facilitated de novo induction of Foxp3+ Treg cells. In this study, we will utilize several approaches including cellular, molecular, biology clinical cohont, and immune- humanized NSG mouse model to investigate the mechanism by which miR-128 regutlates TILs subgroup through regulating cytokines expressed in GC cells. These findings will provide a new theoretical basis for GC immunotherapy

肿瘤浸润淋巴细胞（TILs）具有抑制肿瘤和促进肿瘤生长的双重效应。目前认为TILs促进肿瘤发展与肿瘤分泌特定的细胞因子有关。然而，目前肿瘤-TILs信号的调控机制还不完全清楚。我们的前期研究发现miR-128的异常低表达与CD4+FoxP3+的T细胞亚群增多相关，通过生物信息学分析TCGA数据库发现细胞因子IL16、SEMA4C和STC基因在miR-128低表达的胃癌组织中高表达，机制上我们证实miR-128可直接结合IL16基因3’UTR区域抑制其表达，而IL16可促进Treg表型的T细胞比例增加。在本项研究中，我们将利用包括细胞学、分子生物学、临床队列、人源化免疫小鼠模型方法研究miR-128通过调控肿瘤细胞因子表达影响TILs亚群变化的分子机制，研究发现将为胃癌免疫治疗提供新的理论依据。

背景： .胃癌是全球最常见的恶性肿瘤之一。近年来，尽管胃癌诊治手段有较大突破，但胃癌患者死亡率仍无显著降低，因此，深入探索胃癌发生发展的具体机制，具有重大意义。我们前期开展的研究显示，miR-128-3p在胃癌中低表达，可促进胃癌细胞的增殖。然而，miR-128-3p异常表达是否会影响肿瘤微环境的重塑，从而导致肿瘤免疫逃逸，这一过程仍不清楚。本研究旨在探讨miR-128-3p异常调控肿瘤浸润免疫细胞亚群促进胃癌免疫逃逸的机制。 .方法及内容： .利用生物信息学分析工具分析miR-128-3p在胃癌中的预后意义和miR-128-3p与肿瘤浸润性淋巴细胞的相关性。利用RT-PCR，Western blot及免疫组化分别检测miR-128-3p，相关细胞因子及肿瘤浸润性淋巴细胞表面标志物的表达。荧光素酶报告基因实验证实miR-128-3p的下游靶点。胃癌细胞与淋巴细胞共培养及流式细胞学分析探讨miR-128-3p对肿瘤浸润性淋巴细胞的影响。 .实验结果： .生信分析显示在胃癌中miR-128-3p与大多数肿瘤浸润性淋巴细胞呈负相关性。低表达miR-128-3p与胃癌患者预后差显著相关，并且在CD4+ T cells与Tregs富集时，miR-128-3p低表达预后更差。RT-PCR, Western blot及免疫组化显示miR-128在新鲜胃癌组织中低表达，细胞因子IL16及肿瘤浸润性淋巴细胞表面标志物CD4和Foxp3高表达，并且IL16及Foxp3高表达患者预后更差。荧光素酶报告基因实验，Western blot和ELISA证实miR-128-3p可负性调控IL16蛋白表达及分泌。上调miR-128-3p则相反。此外，胃癌细胞与淋巴细胞共培养及流式细胞学分析显示过表达miR-128-3p通过下调细胞因子IL16减少CD4+CD25+Foxp3+ Tregs的比例，然而下调miR-128-3p后有相反的结果。而且，IL16重组蛋白可促进CD4+CD25+Foxp3+ Tregs的比例。同时针对IL16受体CD4的竞争性抗体可逆转miR-128-3p敲低的效应。这些结果证实在胃癌中miR-128-3p/IL16轴可促进CD4+ Tregs的浸润，这为胃癌的免疫治疗提供了一个很有前景的治疗策略。