CAFs外泌体来源circRNA调控自噬激活促进舌鳞癌化疗耐药的机制研究

Chemoresistance is the main cause for recurrence and poor prognosis in patients with cancer, tumor microenvironment is closely associated with chemoresistance. In the previous study, we confirmed that cancer-associated fibroblasts (CAFs), mainly located in tongue cancer environment, promoted chemoresistance via autophagy. However, the potential mechanism for the activation of autophagy by CAFs in tongue cancer? Our preliminary experiments found that CAFs secreted massive exosomes and exosomes derived circPTBP3（hsa-circ-0003500） activated autophagy and promoted tongue cancer chemoresistance. Bioinformatics tools predicted that there were specific binding sites between circPTBP3 and mir-224-3p and mir-489-3p. Hence, we propose a scientific hypothesis: Cancer-associated fibroblasts exosomes derived circPTBP3 sponge mir-224-3p and mir-489-3p, and upregulates autophagy-related genes contributing to chemoresistance. In this project, we intend to interfere circPTBP3 expression and investigate their function in regulating autophagy activation and chemoresistance in vivo and in vitro models. And we will elucidate the molecular mechanism that exosomes derived circPTBP3 regulates autophagy-related genes. Then we will analyze the relationship between circPTBP3 and clinical features and prognosis in tongue cancer patients. Our research will provide a new target for inhibiting chemoresistance in tongue cancer.

化疗耐药是导致肿瘤复发和患者预后不良的重要因素，肿瘤微环境与化疗耐药密切相关。我们前期工作表明肿瘤相关成纤维细胞（CAFs）是舌鳞癌微环境内的主要细胞类群，能激活舌鳞癌细胞自噬来促进化疗耐药。我们预实验发现，CAF外泌体来源的circPTBP3（hsa-circ-0003500）激活舌鳞癌细胞自噬，促进其化疗耐药。生物信息学软件预测circPTBP3与mir-224-3p和mir-489-3存在特异性结合位点。据此我们提出科学假说：CAFs外泌体来源的circPTBP3竞争性结合mir-224-3p和mir-489-3p，从而上调自噬相关基因激活细胞自噬，促进化疗耐药。本项目拟通过干预circPTBP3，在体内外模型中阐明外泌体来源circPTBP3激活细胞自噬的分子机制，同时分析circPTBP3与舌鳞癌患者临床病理及预后的关系。

在证实circPTBP3调控舌鳞癌化疗耐药及细胞自噬水平之后，在验证circRNA与miRNA的直接结合时，进行RIP、RNA pull down及luciferase实验发现，circPTBP3与生信分析预测的miRNA并没有发生直接性结合。此外，现阶段由于舌鳞癌单独化疗的病例较少，难以收集临床样本，从而影响实验的进行。近年来我们科临床上应用免疫治疗于舌鳞癌逐渐增多，其中靶向PD1/PD-L1免疫检查点的抑制剂在多种肿瘤的治疗上取得了重大突破。然而仍有大量免疫检查点抑制剂的免疫应答病例较低，因此我们将研究内容调整为dCAFs来源外泌体调控口腔鳞癌PD-L1表达及免疫应答机制研究，尝试通过靶向dCAFs来达到增敏OSCC免疫治疗的效果。本课题从CAFs亚群和其分泌的外泌体着手，探究CAFs亚群通过传递分泌外泌体使OSCC中PD-L1表达增高的内在机制，以及其影响免疫治疗效果；进而通过靶向抑制CAFs亚群，减少亚群来源外泌体的分泌，降低OSCC表面PD-L1的表达，起到提高抗PD-1/PD-L1免疫治疗疗效的协同作用。本课题现已证明，CPShigh患者来源的CAFs可通过传递外泌体的方式，上调OSCC表面PD-L1的表达，同时通过临床单细胞数据分析，筛选出特异的CAFs亚群dCAFs，并通过dCAFs来源外泌体转录组测序，鉴定出dCAFs中外泌体所传递进而引起OSCC PD-L1上调的mRNA。本课题目标阐明dCAFs亚群通过传递外泌体引起OSCC细胞内PD-L1表达的具体分子调控机制；探究靶向dCAFs亚群能够增敏免疫治疗效果，有望成为日后协同临床免疫治疗的新靶点和新技术；将dACFs和dCAFs来源的外泌体作为预测免疫治疗疗效的关键因素，具有显著的临床意义。