Atypical Protein Kinase C-? Induces Epithelial Mesenchymal Transition And Immunosuppression Via Snail Signaling Pathway In Cholangiocarcinoma

Part ? Correlation of expressions of aPKC-?,Snail and infiltrated immune cell with clinicopathological characteristics of human cholangiocarcinomaObjectiveTo determine the expression levels of aPKC-?,Snail and immune cells markers such as CD4,CD8,CD25 and Foxp3 in CCA samples.To explore the relationships between aPKC-? and Snail or immune cells markers in CCA tissues.Combined with clinicopathological characteristics of CCA patients,the associations between patient prognosis and expressions of aPKC-?,Snail and infiltrated immune cell were evaluated.MethodsIHC,WB and qRT-PCR were used for examining the mRNA and protein expressions of aPKC-?,Snail and immune cells markers such as CD4,CD8,CD25 and Foxp3 in 64 paired CCA and adjacent non-tumor samples.Clinical correlations were analyzed by the ?2 test and t test.The relationship between aPKC-? and Snail or immnune cells markers in CCA tissues was evaluated by Spearman analysis.The survival among the subgroups was performed with using Kaplan-Meier and log-rank analyses.The univariate and multivariate analyses were assessed by a Cox proportional hazards model.ResultsThe expressions of aPKC-? and Snail were significantly higher in CCA samples than pair-matched non-tumor tissues.The expression of immune markers such as CD4,CD25 and Foxp3 was also higher in CCA tissues compared to pair-matched non-tumor tissues.On the contrary,the expression of CD8 was significantly lower.Low expression of CD8 and over-expression of aPKC-?,Snail,CD4,CD25 and Foxp3 was related to lymph node metastasis,TNM stage ?-? and medium/poorly differentiation in CCA.There was a positive association between aPKC-? and Snail or CD4,CD25,Foxp3 in CCA patient specimens.The Multivariate COX regression analyses indicated that aPKC-?,Snail and immune cell markers were independent prognostic factors on the overall survival of CCA patients.ConclusionThere was a positive association between aPKC-? and Snail in CCA patient specimens.It suggested that aPKC-? involves in CCA epithelial mesenchymal transition.There was a positive relationship between aPKC-? and CD4,CD25,Foxp3 in CCA.aPKC-?,Snail and immune cell markers were independent prognostic factors on the overall survival of CCA patients.Part ? aPKC-? induces epithelial-mesenchymal transition in human cholangiocarcinoma cell linesObjectiveTo explore the regulatory function of aPKC-? in human CCA cell lines epithelial-mesenchymal transition.MethodsTo establish stable human CCA cell lines that up-regulated the expression level of aPKC-? in vitro via transfecting with human aPKC-?-cDNA,WB and qRT-PCR were employed to determine the expression of epithelial markers(E-cadherin and ?-catenin)and mesenchymal marker(N-cadherin).Transwell invasion assay,Wounding heal assay and CCK8 cell proliferation assay were used to detect the ability of invasion,metastasis and proliferation.Then specific aPKC-?-siRNA was transfected into CCA cells with aPKC-?-cDNA.The expression of EMT markers,cell proliferation,invasion and migration were examined.To further examine the effects of aPKC-? in CCA invasion and metastasis in vivo,CCA cells that stably up-regulate aPKC-? were implanted subcutaneous(s.c.)and intravenously(i.v.)into Balb/C nude mice.ResultsAlong with the aPKC-? up-regulation,CCA cell lines showed EMT-like features on mRNA and protein expression and cellular characteristics,including down-regulated epithelial markers E-cadherin and ?-catenin,up-regulated mesenchymal marker N-cadherin,increased cell proliferation,cell invasion and migration as compared with blank and negative control.When silencing aPKC-? in above cells,these cells reversed the EMT-like changes including down-regulated mesenchymal markers,up-regulated epithelial markers,reduced cell proliferation,cell invasion and migration.More tumor cells metastasized to the lungs of nude mice were found in the treatment group.Meanwhile,the volumes of xenograft tumors arising from aPKC-? cDNA transfected CCA cells were larger and more aggressive than in blank and negative control groups.The mRNA and protein expression levels of aPKC-?were significantly increased in the treatment groups as compared to blank and negative control groups.ConclusionsaPKC-? can facilitate CCA invasion and metastasis in vitro and in vivo.These results also suggested that aPKC-? could be an EMT inducer for CCA cells.Part ? aPKC-? induces epithelial-mesenchymal transition and immunosuppression in cholangiocarcinomaObjectiveCombined the Part ? results that there was a correlation between aPKC-? and infiltrated immune cell in CCA,to further investigate the interaction between aPKC-? on regulating EMT and its relationship to CCA immunosuppression.MethodsFresh human PBMCs were first cultured with CCA cells that up-regulate the expression level of aPKC-?(aPKC-?+ CCA cells)for 5 days.CCK8 method was used to detect the proliferation of PBMCs.The number of CD4+,CD8 +cells and CD4+CD25+ cells was analyzed by flow cytometry.After gating CD4+ cells,CD4+CD25+ and CD4+CD25-cells,the percentage of Foxp3+ cells was detected in the culture.CD4+ cells were further isolated from the co-culture system.And then these CD4+ cells were employed to T cell proliferation assay by CCK8 method.When silencing aPKC-? in aPKC-?+CCA cells,the number of CD4+CD25-Foxp3+ cells(Treg-like cells)in co-culture system was measured by flow cytometry.ELISA was employed to examine the immune cytokines IL-2 and TGF-?1 in supernatant of aPKC-?+CCA cells culture.Meanwhile,the number of CD4+CD25-Foxp3-cells was determined in co-cultured system that PBMCs cultured with the supernatant of aPKC-?+ CCA cells culture.Using transwell chambers,PBMCs were separately co-cultured with aPKC-?+ CCA cells and CD4+CD25-Foxp3+ cells was counted.ResultsAfter 5 days co-culture,the proliferation of PBMCs was decreased,and the number of CD4+ and CD8+ cells were decreased as compared to PBMCs cultured with negative control and blank groups.Furthermore,the number of CD4+CD25+ cells including nTreg cells was not increased in the co-culture system.After gating CD4+ cells,CD4-CD25-and CD4-CD25-cells,the percentage of Foxp3-cells was markedly increased in CD4+ cells as compared with those PBMCs cultured with aPKC-?-CCA cells.The CD4+ cells co-cultured with aPKC-?+ CCA cells markedly decreased T cells proliferation,in contrast to the CD4+ cells co-cultured with aPKC-?-CCA cells.Along with the aPKC-? decreasing,the number of Treg-like cells was reduced in co-culture system.The expression levels of IL-2 and TGF-?1 are significantly higher in supernatant of aPKC-?+ CCA cells culture than in supernatant of aPKC-?-CCA cells.The ability to induce immunosuppression including inhibition of PBMCs proliferation,inhibition of T cell proliferation,induction of nTreg-like cells was not changed when PBMCs were separately co-cultured with aPKC-?+ CCA cells using transwell chambers as compared with when PBMCs were co-cultured with aPKC-?+ CCA cells not using chambers.ConclusionsaPKC-i plays an essential role in inducing immunosuppressive Treg-like cells by regulating EMT in CCA cells,partly mediating by Treg-inducible cytokines such as IL-2 and TGF-?1.Part ? Snail is crucial for aPKC-i-induced EMT and immunosuppression in human cholangiocarcinoma cellsObjectiveTo investigate the molecular mechanism of aPKC-i-induced EMT and immunosuppression in human cholangiocarcinoma cells.MethodsThe expression levels of Snail in CCA cells with different expressions of aPKC-? were detected by WB and qRT-PCR.To transfect Snail-siRNA into stable human CCA cell lines that up-regulated the expression level of aPKC-?,WB,IF and qRT-PCR were employed to determine the expression of epithelial markers(E-cadherin and ?-catenin)and mesenchymal marker(N-cadherin).Transwell invasion assay,Wounding heal assay and CCK8 cell proliferation assay were used to detect the ability of invasion,metastasis and proliferation.To transfect Snail-cDNA into stable human CCA cell lines that down-regulate the expression level of aPKC-?,the expression of EMT markers and the ability of CCA cells invasion,metastasis and proliferation were examined.Fresh PBMCs were co-cultured with the CCA cells transfection with aPKC-?-cDNA and Snail-siRNA.CCK8 cell proliferation was used to detect the proliferation of PBMCs.The number of CD4+,CD8-cells and CD4+CD25+ cells was analyzed by flow cytometry.After gating CD4+ cells,CD4+CD25-and CD4+CD25-cells,the percentage of Foxp3-cells was detected in the culture.CD4+ cells were further isolated from the co-culture system And then these CD4+ cells were employed to T cell proliferation assay by CCK8 method.ELISA was employed to examine the immune cytokines IL-2 and TGF-?1 in supernatant of these above CCA cells culture.ResultsWhen increased or decreased the expression of aPKC-? in CCA cells,both protein and mRNA levels of Snail were up-regulated or down-regulated.Followed knockdown Snail in aPKC-?+ CCA cells,the EMT-like changes were almost reversed compared with negative control groups,including the expression levels of EMT markers,the abilities of cell proliferation,invasion and migration.Along with downregulation of Snail expression,the ability to induce nTreg-like cells was significantly decreased in aPKC-?+ CCA cells by Snail-siRNA transfection as compared with negative control groups,as well as promoting PBMCs and T cells proliferation,decreasing immunosuppressive activity of CD4-cells and IL-2 and TGF-(31.Similarly,when Snail was upregulated by transfecting Snail-cDNA in CCA cells with aPKC-siRNA,these cells showed EMT-like features compared with negative control groups,such as decreased epithelial markers and increased mesenchymal markers accompanied by increased cell proliferation,cell invasion and migration.ConclusionsThese findings indicated that Snail is crucial for aPKC-?-induced EMT-like changes and immunosuppression in human CCA cells.Inhibition of Snail could abolish cancer metastasis and immunosuppression.This strategy will provide a new and effective approach to improve the effect of anti-cancer therapy in CCA.