| Pancreatic cancer is one of the most fatal malignant tumors and the fourthcause of cancer-related death. The incidence of pancreatic cancer in China is rising year by year. Like other solid tumors, surgery alone is not the ultimate treatment for this disease. The limited effect and toxicity of standard chemotherapy and radiotherapy make immunotherapy an attractive alternative. However, the immunotherapy is not successful due to the existence of mechanisms of tumor immunologic escape. So the study of the mechanism regarding tumor immunologic escape is of importance.Dendritic cells are the most potent professional antigen-presenting cells (APCs) in the immune system, and they can activate intensive T lymphocyte response against tumors. Tumor cells can evade immune surveillance and establish an immunosuppressive environment that inhibits maturation and function of DC in vitro. Previous studies have found that differentiation and antigen presentation function of DCs are suppressed by pancreatic cancer cell-conditioned medium in vitro;moreover, the number and function of circulating DCs are also impaired in pancreatic cancer patients. But, the detailed mechanisms involved in the effect of pancreatic cancer on DCs remain unclear.MicroRNA (miRNA) is a class of small (20–23nucleotides) endogenous non-coding RNA, which negatively regulates gene expression by inhibiting translation or degradation of mRNA. Furthermore, bioinformatics studies have demonstrated that one miRNA can regulate hundreds of mRNA targets, which could be implicated in almost all physiological and pathological pathways. More recently, studies have indicated that miRNA-146a, a miRNA, can play an important role in m ediating a wide spectrum of biological processes, such as immune and inflammatory response, cell proliferation, differentiation, apoptosis as well as tumorigenesis.Moreover, miRNA-146a has been suggested to be associated with DC differentiation and maturation.As BxPC-3is an extremely metastatic human pancreatic cancer cell line,BxPC-3-conditioned medium (BxCM) was used as pancreatic cancer-conditionedmedium. The aim of this study was to investigate the expression of miRNA-146a inDCs induced by pancreatic cancer cell conditioned medium and its effect on DCmaturation and function.Part1The generation of peripheral blood monocyte-derived DC andthe inhibition of BxCM on the phenotypic maturation of DCObjective: To generate the human peripheral blood monocyte-derived DC. Toinvestigate the inhibitory effect of BxCM on the differentiation and maturation of DC.Methods: Peripheral blood mononuclear cells were obtained from healthy donorsusing lymphoprep. The monocytes were cultured with GM-CSF and IL-4or GM-CSFplus IL-4plus BxCM in vitro. For mature DC, Human TNF-α was added. To study theinhibitory effect of BxCM on the differentiation and maturation of DC, cell surfacemarkers for DC differentiation and maturation were analyzed using flow cytometry.Results: Human CD14+monocytes and DC were successfully generated in vitro.The expression of CD14+in BxCM-treated group were still higher than that in normalgroup (P＜0.05). On the country, the expression of mature phenotypes, such as CD1a,CD80, CD83, CD86and HLA-DR in BxCM-treated group were significantly lowerthan those in normal group (P＜0.05).Conclusion: BxCM could inhibit the differentiation of DC from CD14+monocytesand DC maturation by suppressing the expression of typical phenotype.Part2The effect of BxCM on the expression of miRNA-146a andSmad4protein in DCObjective: To investigate whether the expression of miRNA-146a and Smad4were changed in BxCM-treated DCs. To further determine the role of miRNA-146a onthe expression of Smad4protein. To offer the basic theory for the explaining the mechanism that how DC were inhibited.Methods: QRT-PCR and Western-blot assay were used to investigate the changeof miRNA-146a and Smad4expression in BxCM-treated DCs. To further determine therole of miRNA-146a on Smad4expression, BxCM-treated DCs and DCs weretransfected with miRNA-146a inhibitor or negative control miRNA inhibitor (NCinhibitor) using Lip2000and the expression of Smad4was assessed using QRT-PCRand Western blot assays.Results: The expression levels of miRNA-146a were notably up-regulated byBxCM in DCs compared with normal ones (P＜0.05). However, the protein level ofSmad4in BxCM-treated DCs was significantly lower than those in normal DCs (P＜0.05). Meanwhile, decreased expression of miRNA-146a caused by miRNA-146ainhibitor recovered the reduced expression of Smad4in BxCM-treated DCs and normalDCs (P＜0.05).Conclusion: miRNA-146a negatively regulates the Smad4gene expression both inBxCM-treated DCs and normal DCs. BxCM enhanced the expression level ofmiRNA-146a and inhibited the expression of Smad4, which may contribute to its abilityto suppress maturation and function of DCs.Part3The effects of blocking elevated miRNA-146a in BxCM-inducedDC on the maturation and immune function of DCObjective: To investigate the role of miRNA-146a on BxCM-mediated DCphenotype and immune function change. To study the detailed mechanisms involved inthe effect of pancreatic cancer on DCs and offer the necessary theory forimmunotherapy.Methods: miRNA-146a inhibitor or NC inhibitor were transfected in theBxCM-treated mDCs. The expressions of cell surface markers and IL-12were testedusing flow cytometry and ELISA assay. DCs were loaded with whole tumor cell antigenof BxPC-3cells. The antigen-loaded mDCs were incubated with T lymphocyte cells at aDC/T cell ratio of1:10. Interferon (INF)-γ and IL-12secretion levels were examined at24h after cell mixing using Human IFN-γ ELISPOT Kit and Human IL-12ELISAassay Kit. T lymphocyte proliferation was determined using the MTS assay afterincubation for5days. CTL response was used to investigate the effect of DCs on theactivity of tumor specific CTLs induced in vitro. Results: Inhibition of elevated miRNA-146a significantly increased the expressionof CD1a, CD86, CD80, CD83, and HLA-DR on BxCM-treated DCs (P＜0.05),although still less than untreated DCs. There was a notably increased T cell proliferationrate and stronger CTL response in miRNA-146a inhibitor group compared with that inBxCM-treated group, although still lower than that in normal group (P＜0.05).Conclusion: BxCM reduced phenotypic maturation and the ability of the DCs toactivate the immune response, which is partly through up-regulating miRNA-146aexpression in DCs. |
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