肿瘤外泌体miRNA调控Treg细胞在食管鳞癌放射抗拒中的作用及机制

Esophageal squamous cell carcinoma(ESCC) has poor outcome due to radioresistance. Our previous studies have found that the increasing Treg cells inhibit the killing effect of effector T cells on tumor and secrete cytokines to promote tumor growth, resulting in tumor immune escape during the radiotherapy of ESCC patients. This effect can be increased by the radiation which leads to the tumor resistance to radiotherapy. By inhibiting PTEN, exosomal miR-214/miR-155 could activate the AKT/mTOR pathway to increase the amount and activity of Treg cells, thus inhibiting the effector T cells. DADS activates PTEN which inhibits ATK/mTOR pathway and decreases the ratio of Treg cells. We surmise that exosomal miR-124/miR-155 inhibits PTEN which activates ATK/mTOR pathway to promote the proliferation of Treg cells and inhibit effector T cells, which leads to the tumor resistance to radiotherapy. This project aims to study the mechanism of the exosomal miR-124/miR-155 from ESCC inhibiting PTEN and activating ATK/mTOR pathway to induce the proliferation of Treg cells, and investigate the effects of DADS intervention on radiosensitivity. It is the first time to explore the relationship between exosomes/Treg cells and radioresistance, which is important for the acknowledgement of the mechanism of tumor immune evasion involved in radioresistance and provides new idea for radio-sensitization.

食管鳞癌因放射抗拒而疗效差。课题组发现食管鳞癌患者放疗时Treg细胞增高而抑制效应T细胞杀伤肿瘤细胞的作用，并分泌细胞因子促进肿瘤生长，使肿瘤产生免疫逃逸；放射线可增大此效应使肿瘤抗拒放疗；外泌体miR-214/miR-155可通过抑制PTEN而激活AKT/mTOR通路增加Treg细胞数量及活性进而抑制效应T细胞；DADS激活PTEN而抑制AKT/mTOR通路，减少Treg细胞比率。推测在放疗时外泌体miR-214/miR-155抑制PTEN而激活AKT/mTOR通路促进Treg增殖活化而抑制效应T细胞，导致肿瘤对放射抗拒。本项目拟研究食管鳞癌放疗时外泌体miR-214/miR-155抑制PTEN而激活AKT/mTOR通路进而诱导Treg细胞增殖活化的机制及DADS干预效果。首次探索外泌体/Treg细胞与放射抗拒关系，对了解肿瘤免疫逃逸导致放射抗拒的机制尤为重要，并为放射增敏提供新思路。

放射治疗是食管鳞状细胞癌（ESCC）的主要治疗方法之一，但其疗效受到放射抵抗的限制。microRNA-4443作为近年新报道的miRNA，在多种恶性肿瘤中发挥癌基因的作用，且外泌体miR-4443已被证明参与肺癌的顺铂耐药。本课题旨在探讨肿瘤细胞内及细胞外泌体中miR-4443对ESCC放射敏感性的影响及作用机制。本课题采用分次照射法成功建立辐射抵抗细胞株，并构建miR-4443过表达与及敲减的ESCC稳定感染株，检测细胞增殖、细胞周期与细胞凋亡；X线照射细胞后克隆形成实验检测细胞放射敏感性并检测DNA损伤修复相关蛋白表达。使用加权基因共表达网络分析（WGCNA）和基因集富集分析（GSEA）方法鉴定放射敏感性相关基因及相关通路。实验结果证实，相比于亲本细胞，miR-4443在辐射抵抗细胞及相应的外泌体中显著上调。过表达miR-4443会进一步降低了ESCC细胞的放射敏感性，而抑制miR-4443则增加了ESCC细胞的放射敏感性。miR-4443通过影响DNA损伤修复、凋亡和G2周期阻滞来调节放射敏感性。通过WGCNA和实验验证，我们确定PTPRJ是miRNA-4443调节辐射敏感性的关键靶点。miR-4443过表达或抑制的作用可以通过增加或减少PTPRJ表达来逆转。单基因GSEA分析表明，miR-4443低表达与Epithelial–mesenchymal transition、PI3K-AKT-mTOR、Wnt/β-Catenin等信号通路有关。此外，本课题对Treg细胞进行了分选和培养，并结合免疫数据库分析，结果提示在食管癌中PTPRJ基因的表达与Treg细胞的浸润水平呈正相关。本项目的完成为改善ESCC放射敏感性提供新思路，为逆转ESCC放射抵抗提供新依据。