The Mucin 1 Regulates The Foxp3~+ Treg Cells To Affect The Growth And Metastasis Of Cholangiocarcinoma Through The EGFR-PI3K/AKT Signaling Pathway

Cholangiocarcinoma is a rare heterogeneous malignancy that can be classified as intrahepatic,hilar or distal cholangiocarcinoma based on anatomical location.The treatment methods mainly include surgery,chemotherapy,targeted therapy and immunotherapy,and the clinical treatment of cholangiocarcinoma is included in the form of first-line therapy or combined with second-line therapy.It is worth noting that the tumor microenvironment(TME)plays an important role in the development and invasion of malignant tumors.Tumor-infiltrating lymphocytes are an important component of the adaptive immune system,in which Foxp3~+Treg cells can infiltrate into the cholangiocarcinoma microenvironment to facilitate tumor escape.Therefore,the search for new targets to control the accumulation of Foxp3~+Treg cells in cholangiocarcinoma TME is an urgent problem to be solved.Previous studies have shown that mucin 1(MUC1)can affect tumor cell proliferation,but its mechanism has not been fully elucidated.On the other hand,whether MUC1 can affect the growth and metastasis of cholangiocarcinoma by regulating tumor microenvironment Treg cells,and its mechanism has not been reported.Objective:The purpose of this study was to investigate whether MUC1 affects the activation of EGFR-PI3K/AKT signaling pathway to regulate the proliferation and apoptosis of cholangiocarcinoma through in vitro experiments,and to determine whether MUC1 affects the growth and metastasis of cholangiocarcinoma by regulating Foxp3~+Treg cells in vivo experiments and to explore its possible related mechanisms.Methods:1.Analyze the high-throughput sequencing dataset related to cholangiocarcinoma in the GEO database,screen differentially expressed genes,and intersect with the cholangiocarcinoma-related genes in the Gene Cards and Phenolyzer databases to obtain key targets;The proteins interacting with key targets were retrieved through the STRING database,and the KEGG pathway enrichment analysis was performed to further predict their downstream pathways.2.Human cholangiocarcinoma cell lines and human normal cholangioepithelial cell lines were selected for in vitro cell experiments:CCK-8,scratch and Transwell assays were used to detect cell proliferation,migration and invasion;ELISA was used to detect the levels of proliferation and invasion factors(Ki67,PCNA,MMP-2and MMP-9)and apoptotic factors(Bax,caspase-3 and cleaved caspase-3)in cells.Flow cytometry experiments were used to detect apoptosis;RT-q PCR and Western blot were used to detect the expression of genes and proteins related to MUC1,EGFR and PI3K/AKT pathways.The interaction between MUC1 and EGFR was detected by CO-IP assay.3.CD4~+T cells were extracted and analyzed from normal human peripheral blood,and differentiated into Treg cells were induced by TGFβ1 recombinant protein,and co-cultured with cholangiocarcinoma cells.Flow cytometry was used to analyze the ratio of Foxp3~+Treg cells and the expression of IL-10.4.To construct a mouse model of orthotopic xenograft of cholangiocarcinoma to detect the tumor weight of each group;Immunohistochemical staining was used to detect the expression of MUC1,proliferation and invasion factors(Ki67 and MMP-9).Flow cytometry was used to analyze the proportions of CD4~+T cells,CD8~+T cells,and Foxp3~+Treg cells in mouse tumor tissues.Results:1.Participate in the bioinformatics analysis of key genes for the growth and metastasis of cholangiocarcinomaThe 1926 and 5933 cholangiocarcinoma-related genes obtained from the Gene Cards and Phenolyzer databases,respectively,intersected with the significantly differentially expressed genes in the dataset GSE119336 the GEO database,and 65intersecting genes were obtained,and ESR1,MMP9 and MUC1 were finally determined to be the key genes involved in the growth and metastasis of cholangiocarcinoma.The results of differential analysis of immune invasion based on GSE119336 dataset showed that ESR1 was significantly low in cholangiocarcinoma tissues.MMP9 and MUC1 were significantly highly expressed,and the differential expression of MUC1 was the most significant(log FC=6.337315993).Therefore,the oncogene MUC1 was selected for follow-up studies.2.Effect of MUC1 on the proliferation,migration,invasion and apoptosis of cholangiocarcinoma cellsCompared with HIBEpi C in normal bile duct epithelial cells,the protein expression of MUC1 in three human cholangiocarcinoma cells(RBE,HuCCT1 and Huh28)was significantly increased(P<0.05),and the expression of MUC1 was the highest in HuCCT1 cells.Therefore,HuCCT1 cells were selected for follow-up experiments.Compared with the control group,the expression of MUC1 in HuCCT1 cells in the overexpressing MUC1 group was increased,the cell proliferation,migration and invasion ability were significantly increased,and the expression levels of Ki67,PCNA,MMP-2 and MMP-9 were significantly increased(P<0.05),while those in the knockdown MUC1 group were significantly decreased(P<0.05).The levels of apoptosis,Bax and cleaved caspase-3 in HuCCT1 cells overexpressing MUC1 were significantly decreased(P<0.05),while the apoptosis of HuCCT1 cells in the MUC1knockdown group was significantly increased(P<0.05).3.MUC1 activates the EGFR-PI3K/AKT signaling pathway in cholangiocarcinoma cellsA total of 20 proteins interacting with MUC1 were retrieved from the STRING database,and KEGG pathway enrichment analysis showed that these proteins were mainly enriched in Proteoglycans in cancer,Endocrine resistance,Breast cancer,Focal adhesion and EGFR tyrosine kinase inhibitor resistance and other pathways.The results of Western blot showed that compared with the control group,the expression of EGFR protein and the ratios of p-PI3K/PI3K and p-AKT/AKT in the overexpressed MUC1 group were significantly increased(P<0.05).The knockdown of MUC1 group was significantly reduced(P<0.05).Overexpression of EGFR in MUC1 knockdown cells:Compared with the MUC1 knockdown group,there was no significant change in the protein expression of MUC1(P>0.05),and the protein expression of EGFR and p-EGFR,as well as the ratios of p-PI3K/PI3K and p-AKT/AKT were significantly increased(P<0.05).4.Effect of MUC1 knockdown on Foxp3~+Treg cells in the cholangiocarcinoma tumor microenvironmentThe results showed that B cells naive,T cells regulatory(Tregs),T cells gamma delta,Macrophages M0 and Dendritic cells resting were increased in cholangiocarcinoma tissues.Compared with the control group,the weight of tumors,the positive expression of MUC1 protein in tumor tissues,the proportion of Treg cells,the proportion of Foxp3-positive cells in T cells and the expression of IL-10 in Treg cells were significantly reduced in the MUC1 knockdown group(P<0.05).The proportions of CD4~+T cells and CD8~+T cells were significantly increased(P<0.05).5.Overexpression of MUC1 and knockdown of EGFR on the proliferation,migration,invasion and apoptosis of cholangiocarcinoma cells and Foxp3~+Treg cells in the tumor microenvironmentThe results of transfection and co-culture with Treg cells of HuCCT1 cells showed that compared with the overexpression of MUC1 alone,the overexpression of MUC1 simultaneously reduced the expression of EGFR and p-EGFR proteins,the ratio of p-PI3K/PI3K and p-AKT/AKT,the proliferation,migration and invasion of cells,and the levels of Ki67,PCNA,MMP-2 and MMP-9 in cholangiocarcinoma cells in the EGFR group.The expression of Foxp3 and IL-10 in Treg cells was significantly reduced.The levels of Bax and cleaved caspase-3 were significantly increased,and the number of apoptosis was significantly increased(P<0.05).Compared with the MUC1 overexpression group and EGFR knockdown group,the ratio of p-PI3K/PI3K and p-AKT/AKT in cholangiocarcinoma cells,the proliferation,migration and invasion ability of cells,the levels of Ki67,PCNA,MMP-2 and MMP-9,and the expression of Foxp3 and IL-10 in Treg cells were significantly increased after the addition of PI3K/AKT activator in the MUC1 simultaneous knockdown EGFR group.The levels of Bax and cleaved caspase-3 were significantly reduced,and the number of apoptosis was significantly reduced(P<0.05).6.Effect of MUC1 knockdown with overexpression of EGFR and anti-CTLA-4 in vivo on the proliferation and invasion of Foxp3~+Treg cells and cholangiocarcinoma in the cholangiocarcinoma tumor microenvironmentCompared with the MUC1 knockdown group,the tumor weight,tumor-to-liver weight ratio,EGFR and p-EGFR protein expression in mouse tissues,the ratio of p-PI3K/PI3K and p-AKT/AKT,the expression of Ki67 and MMP-9,and the proportion of Treg cells in the MUC1 knockdown group were significantly increased(P<0.05).The proportion of CD4~+T cells and CD8~+T cells was significantly reduced(P<0.05).Compared with the MUC1 knockdown and overexpression EGFR group,the tumor weight,tumor-to-liver weight ratio,Ki67 and MMP-9 expression in mouse tissues,and the proportion of Treg cells in the MUC1 knockdown MUC1overexpression group were significantly reduced(P<0.05).The proportions of CD4~+T cells and CD8~+T cells were significantly increased(P<0.05).Conclusions:1.MUC1 promotes the proliferation,migration and invasion of cholangiocarcinoma cells,and inhibits the apoptosis of cholangiocarcinoma cells.2.MUC1 can induce the aggregation of Foxp3~+Treg cells in the microenvironment of cholangiocarcinoma tumors and increase the expression of Treg cell immunosuppressive factors.3.MUC1 can affect the apoptosis of cholangiocarcinoma cells by activating the EGFR-PI3K/AKT signaling pathway,and by inducing the aggregation of Foxp3~+Treg cells in the tumor microenvironment,thereby enhancing the malignant phenotype and tumorigenic ability of cholangiocarcinoma cells in vivo,and ultimately promoting the growth and metastasis of cholangiocarcinoma.