| Background:Pancreatic adenocarcinoma (PC) is one of the most malignant gastrointestinal cancers with an overall5-year survival rate of3%-6%and the morbidity is still increasing over the world. In recent years, the mechanism of immune escape of PC was still uncertain and there was little advancement in the immune therapy against PC. The exosomal microRNAs (miRNAs) derived from cancer cells have been proved to be a good biomarker for cancer diagnosis and participate in intercellular communication. Altered expressions of serum miRNAs have been reported to correlate with carcinogenesis and progression of PC, while description of serum exosomal miRNAs and its immune function is still lacking, especially how exosomal miRNA influencing dendritic cell (DC) differentiation and antigen presenting process is still unknown. So it is important to explore new PC related serum exosomal miRNA biomarkers and elucidate the mechanism of pancreatic cancer derived exosomal miRNA influencing DC differentiation and gene expression, which will contribute to early diagnosis and new immune therapy of PC.Objectives:This study was designed for the following objectives:(1) to evaluate serum exosomal miRNAs levels in PC patients, investigating their relationship with clinicopathologic features and prognosis;(2) to demonstrate the mechanism of inhibition of cytotoxic T lymphocyte (CTL) induced by PC exosome and set up new ex vivo model of CTL induction by PC exosome lysate;(3) to elucidate the mechanism of exosomal miRNA influencing DC differentiation and gene expression, exploring the regulating network between the PC derived exosomal miRNAs and DC mRNAs. Methods:Serum exosomal miR-21and miR-17-5p were examined by RT-PCR in a group of49patients, including22cases with PC,6cases with benign pancreatic tumor,7cases with ampullary carcinoma,6cases with chronic pancreatitis, and8healthy subjects. The clinical pathological data were also collected and PC patients were classfied according to the presence of metastasis, tumor differentiation and advanced stage. An ex vivo model was set up to evaluate CTL which were induced by PC exosome and exosome ultrafiltered lysate and their specific killing ability to aim at PC. The expression levels of TNF-a and perforin were examined by western-blot and Elisa in different CTL groups. CCK-8was used to examine the proliferation of CTL and killing rate of CTL for PANC-1. mRNA profiles were examined by chips and differential expression of DC mRNAs were analyzed between immature DC and PC exosome stimulated DC. The target correlations between PC miRNA and DC mRNA were analyzed by internet biological information database.Results:Serum exosomal miR-17-5p and miR-21were higher in PC patients than that in non-PC patients and healthy subjects, with a ROC under curve area of0.887and0.897for diagnosing PC. High level of miR-17-5p was significantly correlated with metastasis and advanced stage. The expression of TNF-a and perforin were inhibited in the CTLs stimulated by PC exosome, which inhibited the specific killing effect of CTL for PC. PC derived exosome lysate followed by ultrafiltration can activate the specific anti-tumor effect of CTL. PC exosome up-regulate12PC related miRNAs in E-DC, which were also highly expressed in PC exosome.4929mRNAs were inhibited in E-DC, including208negative expressed mRNAs. Targets correlations exist between the12PC related miRNAs and208negative expressed mRNAs. RFXAP, the key modulator of MHC â…¡, were negative expressed in DC after stimulation of PC exosome. RFXAP may modulated by4miRNAs (miR-203, miR-101-3pã€miR-212-3pã€miR-139-5p). Conclusions:The exosomal miR-21and miR-17-5p of PC were significantly higher than that of non-PC, which provide significant diagnostic values for PC. Examination of serum exosomal miR-17-5p presents us a useful serum biomarker for PC classification, prognosis and surgical indicator. PC exosome may play important roles in immune escape by down-regulating TNF-a and perforin expression in DC/CTLs. PC exosomal miRNA participates in cancer induced immune escape and the exosome lysate followed by ultrafiltration can activate the specific anti-tumor effect of CTL significantly. PC derived exosomal miRNAs can be transferred into immature DC and influence the gene expression of DC. RFXAP, which regulates the expression of MHC â…¡ molecule, was one of the genes inhibited by PC exosome, which may play important roles in immune escape of PC. We successfully predicted and build the regulating network diagram of the PC derived exosomal miRNAs target relationships with DC mRNA, which provided sufficient directions for exploring the mechanisms of PC derived exosomal miRNAs regulating DC gene expression. |
