miR-191在抗肿瘤CD8+T细胞功能缺陷调控中的作用及其机制研究

The inhibition of anti-tumor CD8+ T cell function by tumor microenvironment is a key factor of tumor immune escape and the illumination of molecule mechanisms of anti-tumor CD8+ T cell dysfunction is very important for the study and development of anti-tuomor immunotherpeutic strategies. In our precious studies, it was found that miR-191 in tumor-infiltrationg CD8+ T cells from human lung cancer tissues is upregulated.The tumor-infiltrating CD8+ T cells from lung cancer tissues of wild-type mice are functionally deficient, with reduction in granzyme B expression. The tumor-infiltrating CD8+ T cells from miR-191 knockout mice have an upregulated granzyme B expression and increased capacity to lyse tumor cells in vitro compared with those from wild-type mice. More importantly,the tumor growth was inhibited in miR-191 knockout mice. These results suggest that miR-191 may be important in regulating the dysfunction of anti-tumor CD8+ T cells. One miRNA can inhibit the translations of many different mRNAs and regulate several functions of one type of cells. Besides granzyme B expression, it is unknown whether miR-191 can also regulate the other functions of anti-tumor CD8+ T cels,such as proliferation, apoptosis, infiltration in tumor tissue, IFN-gamma secretion, and how miR-191 acts. In this study,based on our precious studies, we intend to further investigate the effect of miR-191 on differentiation, proliferation, apoptosis and immune function of anti-tumor CD8+ T cells and to illuminate the molecular mechanisms of miR-191 to regulate CD8+ T cell dysfunction induced by tumor. The aim of this study is to provide experimental data for the intracellular mechanisms of anti-tumor CD8+ T cell dysfunction and provide more alternative targets for anti-tumor immunotherapy based on the enhancement of CD8+ T cell immune function.

肿瘤微环境抑制CD8+T细胞功能是肿瘤逃避免疫监视的重要原因。因此，深入研究抗肿瘤CD8+T细胞功能缺陷的分子机制对肿瘤细胞免疫治疗具有重要的意义。我们的前期研究发现，人肺癌组织CD8+T细胞的miR-191表达被显著上调，敲除小鼠CD8+T细胞的miR-191则可明显提高其颗粒酶表达和肿瘤杀伤活性，并显著抑制肺癌移植瘤的生长，提示miR-191是抗肿瘤CD8+T细胞功能缺陷的重要调控分子。本研究拟在此基础上，以小鼠肺癌为模型，深入研究miR-191对CD8+T细胞的肿瘤组织浸润、颗粒酶释放、穿孔素表达和释放、IFN-gamma等细胞因子分泌、增殖和凋亡等功能的影响，确定受miR-191调控的抗肿瘤CD8+T细胞的功能，并深入研究miR-191调控CD8+T细胞颗粒酶表达等功能的具体分子机制，为抗肿瘤CD8+T细胞功能缺陷的胞内分子机制研究奠定基础，并可能为肿瘤细胞免疫治疗提供新的靶标。

肿瘤微环境抑制CD8+ T细胞功能是肿瘤逃避免疫监视的重要原因，深入研究抗肿瘤CD8+ T细胞功能缺陷的分子机制对肿瘤细胞免疫治疗具有重要的意义。我们的前期研究提示，miR-191是抗肿瘤CD8+ T细胞功能缺陷的重要调控分子，但其对CD8+ T细胞抗肿瘤功能的影响及其具体分子机制尚不清楚。按照项目计划，本研究详细分析了miR-191在抗肿瘤CD8+ T 细胞功能缺陷中的调控作用，初步探索了miR-191调控CD8+ T 细胞颗粒酶B（GZMB）、颗粒酶A（GZMA）、γ干扰素（IFN-γ）、肿瘤坏死因子α（TNF-α）和穿孔素1（PRF1）等分子表达的分子机制。结果发现，与野生型小鼠相比，miR-191过表达荷瘤小鼠的肿瘤生长速度明显加快，miR-191的过表达可显著减少荷瘤小鼠肿瘤组织中浸润的CD8+ T细胞的数量。体外检测发现，miR-191的过表达可显著抑制CD8+ T细胞抗肿瘤活性关键分子IFN-γ、GZMA、GZMB、TNF-α和PRF1等分子的表达，抑制与CD8+ T细胞迁移有关的CCL4和CCR5等分子的表达，抑制CD8+ T细胞向中央型记忆细胞分化，抑制初始CD8+ T细胞的活化后凋亡，而对初始CD8+ T细胞的活化后增殖则无明显影响。进一步的机制研究表明，miR-191过表达不影响T细胞的发育和成熟，miR-191可直接靶向CD8+ T细胞的早期生长反应蛋白2（EGR2）基因，抑制EGR2蛋白的表达，进而下调IFN-γ、GZMA、GZMB、TNF-α和PRF1等分子的表达，导致CD8+ T细胞的功能缺陷。这些研究结果的取得，为抗肿瘤CD8+ T细胞功能缺陷的胞内分子机制研究提供了更多的实验数据，为肿瘤免疫治疗的研究提供了可能的新靶标，也为CD8+ T细胞肿瘤浸润、分化调控等的研究提供了初步的实验数据。同时，我们还发现，miR-191过表达可抑制NK细胞的肿瘤杀伤活性及其活化后凋亡。miR-21的过表达可导致小鼠骨髓中CD8+ T细胞等T细胞数量的显著减少，促进荷瘤小鼠的肿瘤生长，抑制CD8+ T细胞和CD4+ T细胞的肿瘤浸润，并抑制初始CD8+ T细胞的活化后凋亡。这些初步实验结果的取得为后续的深入研究奠定了坚实的实验基础。