胰腺癌诱导DC分化和DC-SIGN表达的microRNA调控机制研究

Pancreatic carcinoma is one of the most maligment tumors with poor 5 years survival rate. The researchs for the immune escape mechanisms and inhibiting methods of pancreatic carcinoma are very valueble for improving biotherapeutic effect and better prognosis of pancreatic carcinoma. We have found that DC-SIGN played important roles in mediating tumor cell adhesion and immune escape, while it is still uncertain about the mutual functions between tumor cells and dendritic cells (DC). This study was designed to explore the mechanisms of miRNA regulating DC-SIGN expression by examining the miRNA profiles of DC, screening with bioinformatics tools and validating the miRNA molecules which regulating DC-SIGN expression; DC-SIGN mediated adhesion between pancreatic carcinoma cell and DC will be performed to study the mutual functions between pancreatic carcinoma cell and DC, demonstrating the molecule mechanisms that how pancreatic carcinoma influencing DC differentiation and DC-SIGN expression by miRNA pathway. By this study, we will proved new theories about pancreatic carcinoma induced immune escape and suggest new methods for inhibiting immune escape and improve biotherapeutic effect of pancreatic carcinoma.

胰腺癌是恶性程度最高、预后最差的恶性肿瘤之一，研究胰腺癌的免疫逃逸机制及抑制方法对探索胰腺癌的生物治疗，改善预后具有重要意义。本课题组前期研究发现DC-SIGN在介导肿瘤细胞粘附与免疫逃逸中发挥作用，但目前对于肿瘤细胞与树突状细胞(DC)间的相互作用的机制仍然不清楚。本项目拟通过检测DC分化过程中microRNA（微小RNA,miRNA）表达谱变化及其与DC-SIGN等DC细胞表型的关系，利用生物信息学工具筛选并验证直接调控DC-SIGN基因表达的miRNA分子，阐明DC分化过程中调控DC-SIGN表达的分子机制；通过DC-SIGN介导的胰腺癌-DC粘附试验，研究胰腺癌细胞与DC间相互作用，阐明胰腺癌通过miRNA途径影响DC分化和诱导免疫逃逸的分子机制，从而为抑制胰腺癌免疫逃逸，提高胰腺癌免疫治疗效果提供新的理论与技术方法。

胰腺癌是临床常见的消化系统恶性肿瘤，预后极差，五年生存率仅为10%，研究胰腺癌免疫逃逸的分子机制及抑制方法，对胰腺癌早期诊断和预后评估有重要意义。本课题组成功从外周血中分离单核细胞，体外诱导未成熟树突状细胞（DC）和成熟DC，利用高通量测序检测DC分化过程中miRNA表达谱变化，并分析差异表达的miRNA与DC-SIGN的关系。利用生物信息学工具和双荧光素酶报告实验筛选并验证靶向DC-SIGN的miRNA，通过细胞荧光标记实验证明DC-SIGN在DC与肿瘤细胞粘附中的重要作用，阐明了miR-186在DC分化过程中调控DC-SIGN从而影响DC分化的分子机制。进一步我们采用超速离心的方法分离胰腺癌外泌体（exosome），通过胰腺癌相关miRNA的qPCR阵列检测实验，发现胰腺癌细胞分泌的exosomal miRNA可以依靠exosome转运入未成熟DC中表达，胰腺癌exosome通过miR-212-3p调控RFXAP表达，并显著下调肿瘤微环境中MHC II类分子表达。同时胰腺癌exosome通过miR-203调控DC TLR4的表达水平，降低肿瘤坏死因子α（TNF-α）及白介素12（IL-12）的水平，继而影响DC的分化和抗原递呈功能。另外，胰腺癌患者的血清exosomal miR-17-5p表达水平对胰腺癌的预后判断以及手术适应征的选择具有非常重要的价值。本课题从分子生物学、细胞生物学和临床医学等多个角度阐明了miRNA在胰腺癌免疫逃逸、早期诊断及预后判断中的作用，为胰腺癌免疫治疗及诊断提供了新的理论与技术方法。