外泌体miR-24调控MDSC促进结直肠癌免疫逃逸的分子机制研究

Our previous study has confirmed that myeloid-derived suppressor cell (MDSC) was amplified in colorectal cancer, which led to tumor immune escape via inhibiting T cell function. And our recent findings have showed that tumor exosomal miR-24 mediated inhibition of T cell function. Nevertheless, it remains unclear whether exosomal miR-24 mediates MDSC to regulate T cell function. Our recent experiment found that tumor derived exosomes promoted MDSC amplification. Furthermore, miR-24 was found to promote MDSC expansion and upregulate the expression of PD-L1. What’s more, the level of exosomal miR-24 was found to be significantly increased in the serum of CRC patients. The present study will illustrate the role of exosomal miR-24 on regulating amplification and function of MDSC via exosomes co-culture experientment and identify the specific molecular mechanism by CRISPR-Cas9 and RNA-sequence, which will be validated by animal model and clinical specimen. This study will elucidate the molecular mechanism of immunoescape that exosomal miR-24 modulates MDSC funciton to inhibit T cell function, which provides novel target and insight for immunotherapy for CRC

申请人前期研究发现髓系来源抑制细胞（MDSC）在结直肠癌中扩增并抑制T细胞的功能，从而促进肿瘤免疫逃逸；且肿瘤外泌体通过介导miR-24抑制 T细胞功能；但外泌体miR-24是否通过MDSC调控T细胞功能及其分子机制尚不清楚。申请人新近研究发现：外泌体促进与其共培养的MDSC扩增；进一步研究发现外泌体miR-24可促进MDSC扩增并上调PD-L1的表达；且外泌体miR-24在结直肠癌患者血清中上调。本项目拟：通过外泌体共培养实验明确外泌体miR-24在调控MDSC扩增和功能中的作用；再利用CRISPR-Cas9和RNA-seq技术鉴定外泌体miR-24调控MDSC的具体分子机制；并在动物模型及临床样本中验证分子机制。本项目的完成，将阐明外泌体miR-24调控MDSC扩增与功能从而抑制T细胞功能，促进结直肠癌免疫逃逸的分子机制。进而发现新的免疫负调控靶点，为结直肠癌免疫治疗提供新的思路。

恶性肿瘤是危害我国人民健康的重大疾病，已有研究证实肿瘤存在免疫逃逸，在前期研究中我们发现髓系来源抑制细胞（Myeloid-derived suppressor cell, MDSC）在肿瘤微环境中大量积累，并抑制肿瘤杀伤细胞NK细胞及CTL细胞的功能，促进免疫逃逸和转移，然而其机制目前尚未清楚。我们前期研究发现提示外泌体在肿瘤微环境免疫负调控中具有重要作用，因此本项目主要探究外泌体对MDSC的影响及其分子机制。本项目首先建立了外泌体研究平台，对外泌体的分离、纯化、鉴定、表征做了系列的证明。在此基础上分离肿瘤来源的外泌体，通过功能实验发现外泌体可以促进MDSC的扩增。进一步通过对miR-24敲低实验发现miR-24在外泌体介导MDSC扩增中起着重要作用。结合生物信息学预测及功能实验发现miR-24通过结合STING的3’UTR方式调控STING的表达,进一步通过体内外功能实验证实miR-24的下游STING阻断了MDSC的扩增。为STING抑制剂应用在临床肿瘤免疫治疗上提供了理论基础和新的思路。