Pathogenesis Of Promotion On Pancreatic Cancer Cells Growth And Inhibition On Tumor Immunity Induced REG3A

Objective To investigate the mechanism of regenerating gene protein(REG) 3A on the cell proliferation,so as to confirm the combination of REG3 A and EGFR to pancreatic cancer cell(PaC cell) growth. Under the inflammation environment induced by IL-6, REG3 A was found to mediate the genesis and development of PaC via JAK2/STAT3 signalling pathway. Futhermore, to explore the immunological mechanism of Reg3g(a mouse homolog of human REG3A) overexpression on the tumorigenesis, and to explicit the function of DCs and CD8+T cells, the key target cell regulated by Reg3 g overexpression, Providing new ideas for the research on the pathogenesis and drug therapeutic targets of pancreatic cancer.Methods Firstly, a pancreatic cancer cells line were used in the study, SW1990 and BxPC-3, whose were treated with REG3 A siRNA, cell proliferation was assayed by MTT method and cell cycle and apoptosis were analyzed by fluorescence-activated cell sorting(FACS). Real-time PCR and western blot was used for the determination of JAK2, STAT3, C yclinD1, Bcl2, REG3 A expression. Further investigation on the inhibition of JAK2, STAT3 was used to study JAK2/STAT3/REG3 A positive feedback loop. Secondly, immunoprecipitation were used to clarify the combination of REG3 A and its receptors, which then activated JAK2/STAT3 signalling pathways to promote the PaC growth.Simultaneously, BxPC-3 cells were incubated with different dose of IL-6, we found that IL-6 stimulation induced REG3 A expression via a concentration-dependent mechanism. We next incubated SW1990 with siRNA-STAT3 for 24 h, in order to explore whether or not IL-6 activated JAK2/STAT3 signalling pathways to promote the expression of REG3 A. The differential expressed genes and key nodes in SW1990, induced by enhanced REG3 A or IL-6 were determined by Microarrary. The mechanism of pro-tumor effect was exerted by REG3 A in synergism with IL-6 in nude mice xenografted with PaC cells. Finally, to explore the immunological mechanism of Reg3 g on the tumorigenesis, we investigated the secretion of TGF-β, IL-10, IFN-γ and IL-12P70 of dendritic cells treated with exogenous Reg3 g by ELISA. The expression of DCs marker CD11 c, MHC-II and the co-stimulatory molecule CD86 were analyzed by FACS when DCs were treated with exogenous Reg3 g or conditioned media from Panc02 tumor cells in vitro for 24 h. C57BL/6 mice were inoculated subcutaneous in the left flank with Panc02 cells(2×105 cells) in serum- free RPMI 1640 medium. Five day to induce tumorigenicity and then injected with pReg3 g, a lentivirus system to stimulate production of murine Reg3 g, or shReg3 g, a lentivirus system to inhibit production of murine Reg3 g, respectively. After two weeks, mice were sacrificed and tumors were dissected. Analyses included assessment of pancreatic and spleen histopathological characteristics, serum biochemical indicators, the ratio of CD8+T cells, Tregs, MDSCs, DCs marker CD11 c, MHC-II and the co-stimulatory molecule CD86, migration and phagocytotic functions on DCs, quantification of tumor-related gene expression, tumorigenicity-associated pathway activation, and the expression of inflammatory cytokines and proliferation-associated gene.Results Firstly, we applied siRNA to silence STAT3 and REG3 A or AG490 to block JAK2 phosphorylation, a significant decrease was observed in the expression of REG3 A, CyclinD1, Bcl2 and the proliferation of PaC, suggesting JAK2/STAT3 pathway was involved in REG3 A self- induction. Meanwhile, REG3A/EGFR complexes were measured by IP, and pre-treatment with EGFR-specific inhibitor results in a significant decreased expression of pSTAT3, REG3 A in PaC cells. REG3A/EGFR complexes induction through JAK2/STAT3/REG3 A pathway created a positive feedback mechanism to further increase REG3 A expression thus amplifying its pro-growth effect. The expression of REG3 A was enhanced by IL-6 stimulation in BxPC-3. When we applied siRNA to silence STAT3 phosphorylation, IL-6 could not upregulate the expression of REG3 A through JAK2/STAT3 signalling pathways. Furthermore, the Microarray analysis showed that both of REG3 A and IL-6 might share functional interrelationship networks especially linking cell growth-association signaling pathways. The similar nodes of STAT family in the networks were regulated by REG3 A or IL-6. REG3 A in synergism with IL-6 exerts a marked pro-tumor effect in vitro and in vivo. Finally, Reg3 g inhibited the maturation status and migration phenotype of DCs, and reduced the secretion of IL-12, IFN-γ, while enhancing the secretion of IL-10, TGF-β. What is more, Reg3 g supressed the number of CD8+T cells infiltration in local tumor tissure, accompanying by upregulation the negative regulator of C D152, PD-1, PD-L1 and Tregs. We silenced C D8+T cells with blocking antibody(anti-CD8) and silence of Reg3 g could not significantly abolish the increase of tumor volume induced by anti-CD8 effects. Further study inveategated that Reg3 g promoted tumor growth and inhibited the activation of T cell responses by DCs and CD8+T cells via STAT3 signaling pathway, meanwhile Reg3 g promoted the pancreatic cancer initiation and development through activating the STAT3 pathway to upregulate the the expression of K i67 and lower the expression of SOCS3, caspase3.Conclusion These results suggest IL-6 combines with the IL-6R and gp130, activates the downstream JAK2/STAT3, enhancing the expression of REG3 A. REG3 A bonds with EGFR, promotes self-autocrine through a similar pathway to constitute a REG3A-JAK2/STAT3 positive feedback loop. Further investigation manifest that REG3 A exerts a marked pro-tumor effect in vitro and in vivo under the inflammation environment induced by IL-6. Reg3 g inhibites the maturation of DCs and the cross-priming between C D8+ T cells through activating the STAT3 pathway in DCs, while Reg3 g promotes the number of Tregs in coordination with enhancing production of Th2 cytokines to suppress anti-tumor immune responses. Subsequently, we silence CD8+T cells with blocking antibody(anti-CD8), Reg3 g reduces the tumoricidal activity of CD8+T cells via inhibiting STAT3 pathway in CD8+T cells. Meanwhile, Reg3 g accelerates pancreatic cancer initiation and development through activating STAT3 pathway in tumor cel s. Innovative points 1. Our study first provides the proof that REG3 A initiates tumorogenesis and a REG3A-JAK2/STAT3 feedback loop-associated mechanism under the inflammation environment induced by IL-6. 2. The proposed project for the first time employs orthotopic mouse pancreatic carcinoma model to clarify that Reg3 g weakenes immunological mechanism on the tumorigenesis through inhibiting DCs maturation, the frequency of C D8+T cells and the cross-priming ability of DCs and CD8+T cells.