结直肠癌中miR-195调控PD-L1的表达和生物学功能的研究

Colorectal cancer is one of the frequently occurred cancers in human, the fourth cause of death leading in worldwide. The rapid proliferation of tumor cells needs to build their own tissue environment, named tumor microenvironment. Recently, the immunosuppression mediated by tumor microenvironment molecules received extensive attention in tumor immune research. PD-L1, one of the B7 families was cloned form the human DC cDNA library in 1999. Programmed death ligand 1 (PD-L1) is a 40kDa type 1 transmembrane protein and located in Chromosome 9p24.2. The mRNA of human PD-L1 is generally expressed in the human tissues, while the protein is rarely expressed in the normal tissues. The group of applicant has accumulated a large amount research achievement in PD-L1 and B7 gene family research. We found that PD-L1 highly expressed in colorectal cancer tissues and was correlated with prognosis of patients previously. On this basis, we plan to search the regulation mechanism of PD-L1. We performed the microRNA array of clinical colorectal cancer tissues and screened differentiated microRNA which predicted to binds to 3’-UTR of PD-L1 by software. We will further confirm the microRNA and PD-L1 expression in fresh tumor tissue. Next, we will verify that miR-195 binds to the 3’-UTR of PD-L1 by luciferase assay and regulates protein expression and biology function of PD-L1 via in vitro and in vivo analysis. At last, we put forward the plan of combined therapy by combining miR-195 and PD-L1 antibody. In general, the achievement of this program will improve the regulation mechanism research of PD-L1 and provide the new target for clinical tumor immunotherapy.

负性共刺激分子PD-L1是目前肿瘤免疫逃逸机制研究和免疫治疗的热点，但PD-L1在肿瘤免疫微环境中异常表达的机制尚待阐明。我们前期的研究发现：PD-L1在结直肠癌组织中异常表达，并与病人的预后密切相关，同时利用临床结直肠癌组织进行了miRNA芯片检测与分析，筛选出调控PD-L1表达的差异miRNA--miR-195，并初步证实miR-195参与PD-L1的表达调控。本项目以此为切入点在临床标本及结直肠癌细胞系中进一步分析PD-L1与miR-195的表达及相关性，并利用临床肠癌组织标本、细胞株及模型动物进行体内外实验论证miR-195对PD-L1的表达调控及其分子机制，最后验证miR-195干预与PD-L1抗体对结直肠癌的联合治疗效果。本项目的研究将从microRNA这个新视点阐明负性免疫调控分子PD-L1在肿瘤微环境异常表达的机制，也为PD-L1/PD-1肿瘤免疫治疗提供新的思路。

负性共刺激分子PD-L1是目前肿瘤免疫逃逸机制研究和免疫治疗的热点分子，针对PD-L1的阻断性抗体、伴随诊断抗体研究在近几年有了众多突破性进展，但PD-L1在肿瘤免疫微环境中异常表达的机制仍有待进一步阐明。本课题中我们利用临床结直肠癌组织进行了miRNA芯片检测与分析，筛选出调控PD-L1表达的差异miRNA--miR-195，以此为切入点在新鲜肠癌组织以及结直肠癌细胞系中初步验证两者的表达负相关性；接着通过免疫组化以及原位杂交法检测临床结直肠癌病人的PD-L1和miR-195的表达，统计学验证了两者表达的负相关性；通过临床预后分析，发现miR-195以及miR-195与PD-L1的组合比PD-L1表达能更好的预测病人预后；接下来我们通过luciferase证实了miR-195对PD-L1的直接调控并在多种细胞、不同条件下进行了验证；同时我们也证明了miR-195对细胞增殖凋亡的功能影响是通过调控PD-L1实现的；最后我们通过将miR-195和PD-L1阻断性抗体联合治疗免疫缺陷小鼠验证了miR-195确实可以协同增强PD-L1的治疗效果。本项目通过临床肿瘤组织标本、结直肠癌肿瘤细胞系和免疫缺陷小鼠模型，采用microRNA 芯片、Real time PCR、流式细胞术等手段，从分子、细胞、组织以及动物水平等层次研究了明星分子PD-L1的microRNA调控机制，提出并验证了miR-195联合PD-L1的抗体治疗结直肠癌的新思路，对结直肠癌的免疫治疗提供了新的思路和新的方向，具有潜在的临床应用价值。