Phosphorylation Of Sp1 At Ser59 Site Induces APKC_-1/snail-mediated Epithelial-mesenchymal Transition And Immunosuppression In Cholangiocarcinoma

Part I Quantitative i TRAQ-based phosphoproteomic analysis identify Sp1 as direct phosphorylation substrate for a PKC-ιObjective Identifing the direct phosphorylated substrates and sites for a PKC-ι,and exploring the regulatory mechanism of a PKC-ι in the process of epithelial-mesenchymal transition and immunesuppression in human CCA cell lines.Methods Establishing stable human CCA cell lines that up-regulating the expression level of a PKC-ι in vitro via transfecting with human a PKC-ι-c DNA as treatmental group.The cells with negative virus was as control group.Then we use quantitative i TRAQ-based phosphoproteomic analysis to identify differentilally expressed phosphorylation sites,peptides and proteins,and do the bioinformatics analysis such as GO,KGEE and protein interaction network analysis.Then we select the differentilally expressed phosphorylation protein in the EMT signaling pathways,and vertify the expression by the WB and immunefluorescent(IF).Co-immunoprecipitation was used to detect the mutual combined relationship between this phosphorylation protein and a PKC-ι,and demonstrate that whether t was the direct phosphorylation substrate for a PKC-ι in human CCA tissues and cell lines.Results Quantitative phosphorylation of proteomics demonstrated that in CCA cell lines with a PKC-ι-c DNA,311 phosphorylated peptides,corresponding to 247 phosphorylated proteins,were identified on the basis of special statistical standards(Fold Change≥1.2 and P value≤0.05).Among these 311 phosphorylated peptides,169 were up-regulated and 142 were down-regulated.After bioinformatics analysis,we found that the expression of phosphorylated Sp1 at Ser59 site involved in the TGF-β signaling pathway of EMT was markedly up-regulated after the CAA cells were transfected with a PKC-ι-c DNA.To confirm the role of a PKC-ι in Sp1 phosphorylation at Ser59 site(P-Sp1),WB and IF were used to confirme that Sp1 and P-Sp1 were up-regulated along with the overexpression of a PKC-ι.To investigate whether Sp1 might be a direct substrate of a PKC-ι,we performed Co-IP experiment and found a PKC-ιphysically interacted with Sp1.Conclusion a PKC-ι mediates the phosphorylation of a PKC-ι at Ser59 sites,and Sp1 Ser59 was the direct phosphorylation site for a PKC-ι in CCA.Part II Correlation of expressions of P-Sp1 and a PKC-ι/Snail,infiltrated immune cell with prognosis of human cholangiocarcinomaObjective Combining with the clinical data to exploring the relationship between the expression of P-Sp1 and a PKC-ι/Snail,infiltrated immune cell in holangiocarcinoma.Methods The 64 paired CCA and adjacent non-tumor samples were obtained from January in 2012 to March in 2016.IHC and WB were used to examine the levels of Sp1,P-Sp1,a PKC-ι,Snail and immune cell markers such as CD4,CD8,CD25 and Foxp3.The relationship was evaluated between the expression of P-Sp1 and the other markers by Spearman analysis.The correlations between clinicopathological characteristics(TNM stage,differentiation,lymph node metastasis)and P-Sp1 were analyzed by the χ2 test and T test.The Kaplan-Meier analyses was used to demonstrate the relationship of Sp1,P-Sp1 with the prognosis among these CCA patients.The multivariate analyses were assessed by a Cox proportional hazards model to identify the prognostic role of P-Sp1 for the patients with cholangiocarcinoma.Results The expression of Sp1,P-Sp1,a PKC-ι,Snail and CD4,CD25,Foxp3 were significantly higher in CCA samples than in pair-matched non-tumor tissues,while the CD8 was opposite.In CCA tissues,the level of P-Sp1 was positively correlated with the a PKC-ι,Snail,CD4,CD25,Foxp3 expression,and negatively correlated with the CD8 expression.High expression of Sp1 and P-Sp1 is closely related to clinicopathological characteristics of cholangiocarcinoma.When the expression of Sp1 and P-Sp1 is higher,the differentiation degree is lower,the level of TNM stage is higher,the rate of lymph node metastasis is higher for CCA patients.The patients with high level of Sp1 and P-Sp1 displayed a shorter overall survival compared to those with low expression.Multivariate COX regression analyses indicated that Sp1,P-Sp1 were dangerous factors of the prognosis for the patients with cholangiocarcinoma.Conclusions There was a positive association between P-Sp1 and a PKC-ι/Snail in CCA atient specimens.The expressions of P-Sp1 and CD4、CD25、Foxp3 were also correlated positively,showing that the phosphorylation of Sp1 at Ser59 was associated with the infiltration of immune cells.Sp1,P-Sp1 were dangerous factors of the prognosis for the patients with cholangiocarcinoma.Part III P-Sp1 induce the epithelial-mesenchymal transition through regulating the transcription of Snail in cholangiocarcinomaObjective To demonstrate the role and mechanism of a PKC-ι-mediated Sp1 phosphorylation at Ser59 site regulating the expression of Snail and epithelial-mesenchymal transition in cholangiocarcinoma.Methods we constructed wild-type Sp1(WT-Sp1),Ser59 non-phosphorylatable mutant SP1(S59A-Sp1)and Ser59 phosphomimetic mutant Sp1(S59D-Sp1)eukaryotic expression vectors and generated CCA cells that stably expressed these constructs by amino acid mutant technology.WB、q RT-PCR and IF were used to detect the expression of Snail in these CAA cell lines.Co-IP was used to detect the interaction relationship between Sp1 and Snail in CCA cells and tissues.Chromatin immunoprecipitation(Ch IP)was performed to detect the the binding degree between Sp1 and Snail promoter.WB,q RT-PCR and IF were employed to detect the expression of markers about epithelial-mesenchymal transition in these CCA cells.Transwell invasion assay,Wounding heal assay and CCK8 cell proliferation assay were used to detect the ability of invasion,metastasis and proliferation.Then we conduct lung metastasis model in nude mice to detect the role of Sp1 phosphorylation at Ser59 site on regulating the ability of invasion,metastasis in CCA cells.ResultsThe exprssion of Snail was higher in S59D-Sp1 cells compared with WT-Sp1 cells,while lower in S59A-Sp1 cells.Co-IP was performed to indicate that Sp1 interacted directly with Snail,and Ch IP indicated that the Sp1 phosphorylation at Ser59 site enhanced the enrichment of the immunoprecipitated Snail promoter region with Sp1.S59D-Sp1 cells have higher levels of N-cadherin and lower levels of E-cadherin and β-catenin,whereas S59A-Sp1 cells displayed the opposite expression patterns compared with WT-Sp1 cells.Furthermore,S59D-Sp1 cells exerted potent effects on promoting the ability of progression,migration and invasiveness in CCA cells,whereas S59A-Sp1 cells showed the opposite effects compared with WT-Sp1 cells.The model of lung metastasis demonstrated that more tumor cells metastasized to the lungs of nude mice were found in S59D-Sp1 cells group,while less in S59A-Sp1 cells group.IHC and WB demonstrated that the expression of P-Sp1 in lung metastasis tissues was higher in S59D-Sp1 cells group.Conclusions Sp1 phosphorylation at Ser59 site promote the expression of Snail and epithelial-mesenchymal transition through enhancing the binding of Sp1 to Snail promoter.Part IV P-Sp1-mediated epithelial-mesenchymal transition induces immunosuppression in human cholangiocarcinoma cellsObjective To investigate the role and mechanism of P-Sp1-mediated EMT regulating the number and function of immune cells in human CCA.Methods Establishing human CCA cell lines with EMT-like changes by transfecting the ector with differential expression of P-Sp.The Human Peripheral Blood Mononuclear Cells(PBMCs)were extracted from healthy volunteers.Fresh human PBMCs were first co-cultured with CCA cells with differential expression of P-Sp.After 5 days.CCK8 experiment was used to detect the proliferation of PBMCs.Then we used the flow cytometry to detect the number of CD4+ and CD8+ cells,and detect the percentage of Foxp3+ cells in CD4+,CD4+CD25+ and CD4+CD25-cells.We used the immunomagnetic beads to isolate the CD4+ cells from the co-culture system.And then these CD4+ cells were co-cultured with fresh CD4+ and CD8+ T cells from PBMCs.After 5 days,CCK8 experiment was used to detect the proliferation of CD4+,CD8+ T cells.We also used the transwell chambers to make PBMCs separately co-cultured with CCA cell lines with EMT-like changes,and using the supernatant of the CCA cells with EMT-like changes to culture the PBMCs,then we detected the proliferation of PBMCs,number of CD4+ and CD8+ cells,the percentage of Foxp3+ cells.ELISA was employed to detect the level of immune cytokines IL-2 and TGF-β1 in supernatant of CCA cell lines with EMT-like changes by transfecting the vector with differential expression of P-Sp1.Results The proliferation of PBMCs was decreased,and the number of CD4+ and CD8+cells were also decreased in the co-culture system with S59D-Sp1 cells compared to WT-Sp1 cells,while opposite role in the co-culture system with S59A-Sp1 cells.The percentage of Foxp3+ cells in CD4+ cells and CD4+CD25-cells was markedly increased in the co-culture system with S59D-Sp1 cells compared to WT-Sp1 cells,while decreased in the co-culture system with S59A-Sp1 cells.The CD4+ cells isolated from co-cultured system with S59D-Sp1 cells could enhance the ability of inhibiting the proliferation of T cells,while decreased for the CD4+ cells in the co-cultured system with S59A-Sp1 cells.The results were the same in the separately co-cultured system r the PBMCs cultured with the supernatant of the CCA cells with EMT-like changes as mentioned above.ELISA demonstrated that the S59D-Sp1 cells secrete more immune cytokines IL-2 and TGF-β1 than WT-Sp1 cells,while the less in S59A-Sp1 cells.Conclusions In the EMT-model mediated by P-Sp1,the Sp1 phosphorylation at Ser59 site affects the number and function of immunosuppressive CD4+CD25-Foxp3+cells by regulating the secretion of immune cytokines IL-2 and TGF-β1.