结肠癌外泌体运载miR-208b诱导Tregs分化促进肿瘤免疫逃逸的研究

Tumors have the capacity to escape from immune surveillance by numerous mechanisms. One of which is an increase in the population of regulatory T cells (Tregs). Tregs are increased during tumor progression in colon cancer, but the precise mechanism remains poorly understood. Studies indicated that exosomes derived from tumor cells can promote the differentiation of T cells to Tregs, and miRNAs which are key components in the exosomes are important for the maintenance of Tregs homeostasis. In our previous studies, miR-208b was found to be probably related with the immune evasion of colon cancer patients. We also found that miR-208b was sufficiently delivered into CD4+T cells and the percentage of CD25+Foxp3+T cells was obviously increased when CD4+T cells was incubated with SW-480-derived exosomes together. Target prediction and validation assay indicated that PDCD4 was the target gene of miR-208b. In this proposal, we will further verify whether exosome-delivered miR-208b regulates the differentiation of Tregs to promote colon cancer immune evasion. We will also explore the important role of PDCD4 gene in this process. Our study will not only reveal a novel mechanism of immune evasion in colon cancer, but also provide a new direction and a target for colon cancer targeting therapy.

调节性T细胞（Tregs）可以促使肿瘤细胞发生免疫逃逸。结肠癌患者体内Tregs增多，但是增多的机制仍不明确。研究表明肿瘤细胞分泌的外泌体可以促使T细胞向Tregs分化。miRNA是外泌体运载的重要成分，并且已经证实某些miRNA与Tregs的生成密切相关。我们前期研究发现循环miR-208b可能是转移性结肠癌患者免疫逃逸的重要调节因子，进一步分析发现结肠癌外泌体可以运载miR-208b进入CD4+T淋巴细胞，诱导CD4+T淋巴细胞向CD25+Foxp3+T淋巴细胞分化。靶点预测与验证实验初步证实T淋巴细胞中的PDCD4基因为miR-208b的靶基因。本研究旨在原有研究的基础上，进一步验证结肠癌外泌体中的miR-208b能否诱导Tregs生成促使肿瘤免疫逃逸，并探讨PDCD4基因在这一过程中的作用。这对解释结肠癌免疫逃逸的机制有重要的意义，更为结肠癌靶向治疗提供新的方向和靶点。

晚期结直肠癌患者多数接受奥沙利铂等药物为主的化疗方案。但临床治疗中，部分病人表现出对奥沙利铂耐药或反应不敏感。因此，了解化疗过程中所产生的耐药机制，并且寻找可以预测结直肠癌化疗耐药的特异性血清标志物就成了亟待解决的临床问题。微小核糖核酸（microRNA）是长度在22 bp左右的微小非编码RNA，近年来，外泌体来源的miRNA被认为可用作预测化疗敏感性的潜在生物标志物。调节性T细胞（Tregs）以其免疫抑制作用而闻名，靶向Tregs是提高化学敏感性的有效方法。本课题着眼于血清microRNA在预测结直肠癌患者奥沙利铂耐药中的潜在价值和应用，寻找可以用于指导临床化疗方案选择的血清标志物，并将探讨特异性的microRNA在介导奥沙利铂耐药中的分子机制。本研究收集接受FOLFOX治疗的结直肠癌患者的化疗前血清，记录患者化疗效果，通过使用TaqMan Low Density Array从血清中筛选差异表达的血清miRNA，使用RT-qPCR对其进行验证。通过连续差速超速离心分离外泌体，并通过透射电子显微镜对其进行验证。实时定量PCR和蛋白质印迹确定RNA和蛋白质水平。结直肠癌细胞系中miR-208b以外泌体的形式由肿瘤细胞分泌，奥沙利铂耐药细胞表现出明显的miR-208b表达上调现象，这些结肠癌细胞分泌的miR-208b被递送到受体T细胞中，通过靶向程序性细胞死亡因子4（PDCD4）来抑制T细胞中PDCD4基因的表达，诱导T细胞朝Tregs分化，Tregs比例增加。此外，体内研究表明，癌细胞分泌的miR-208b介导的Treg扩增导致肿瘤生长和奥沙利铂耐药。我们的结果表明，耐药细胞来源的外泌体miR-208b通过靶向PDCD4促进Tregs扩增，并降低奥沙利铂的化疗敏感性。本课题为预测结肠癌患者奥沙利铂耐药提供了潜在的血清miRNA标志物，并阐明了miR-208b在诱导癌细胞免疫逃逸中的分子机制，具有重要的临床意义。