Study On The Function And Mechanism Of 14-3-3? In The Regulation Of Malignant Biological Characteristic And Ferroptosis In Cholangiocarcinoma

Part I Construction of a prognostic model for Cholangiocarcinoma based on Ferroptosis-related genesObjective Cholangiocarcinoma(CCA)is a highly malignant disease with extremely lethal.Ferroptosis is a novel regulated form of cell death induced by iron-dependent lipid peroxide accumulation.Targeting ferroptosis has emerged as a potential therapeutic strategy for tumors.The aim of this study was to screen out differentially expressed ferroptosis-related genes(FRGs)in CCA by analyzing the Cancer Genome Atlas(TCGA)database and Ferr Db dataset,and construct a prognostic signature for cholangiocarcinoma.Methods Differentially expressed ferroptosis-related genes(FRGs)were identified by downloading data from the TCGA database and Ferr Db.And functional enrichment analysis and protein-protein interaction analysis were performed for FRGs.Then,the least absolute shrinkage and selection operator(LASSO)Cox regression and stepwise multiple Cox regression were applied to construct a signature and we assessed the independent prognostic value of the signature using Kaplan-Meier survival curve analysis,ROC curves,univariate and multifactor Cox regression analysis.Moreover,we analyzed the correlation between the prognostic signature and immune cell infiltration.Results A total of 152 differentially expressed FRGs were identified in this study.Among them,127 genes were overexpressed and 25 genes were under-expressed.The GO and KEGG enrichment analysis of the above differential genes was utilized to further clarify the functional significance of FRGs in CCA.Then,a signature consisting of three FRGs(BNIP3,GPT2,YWHAE)was successfully constructed by the LASSO regression and stepwise multivariate Cox regression.Moreover,Kaplan-Meier survival curve analysis and ROC curve analysis showed that the signature had good sensitivity and specificity in predicting OS of CCA patients.In addition,univariate and multivariate Cox regression analysis indicated the independent prognostic value of the signature.Further studies revealed that the risk scores of the FRGs signature were positively correlated with dendritic cells,M1 macrophages and neutrophil infiltration,while negatively correlated with plasma cells and CD8+ T cells.Conclusions In summary,we were the first to identify differentially expressed FRGs in CCA and constructed a prognostic signature of three FRGs(BNIP3,GPT2,YWHAE).This signature can potentially assess the overall survival of CCA patients and targeting ferroptosis may be a therapeutic alternative for CCA.Part ? Study on relationship between 14-3-3? and biological characteristics of cholangiocarcinomaObjective The role of 14-3-3? encoded by the YWHAE gene in cholangiocarcinoma(CCA)is still unclear.To detect the expression of 14-3-3? in CCA and its correlation with clinicopathological features,and to explore the effect of 14-3-3? on the malignant biological behavior of CCA.Methods Immunohistochemistry,Western Blot and real time PCR were employed to detect the level of 14-3-3? in CCA tissues and paracancerous tissues.And the relationship between 14-3-3? and the pathological differentiation,lymph node metastasis and TNM stage of CCA was evaluated by the ?2 test.The Kaplan-Meier analysis was used to assess the relationship between 14-3-3? expression and prognosis of patients with CCA.Then,the univariate and multivariate Cox regression analysis were used to value the independent prognostic role of 14-3-3? in CCA.Moreover,the lentiviral technology was applied to construct with stably 14-3-3? overexpressed and downexpressed cholangiocarcinoma cell lines,and the relationship between 14-3-3? and the biological behavior of CCA was explored by in vitro and in vivo experiments.Results The expression level of 14-3-3? was significantly higher in CCA tissues than paracancerous tissues.High level of 14-3-3? was closely related to lymph node metastasis,stage and tissue differentiation.Kaplan-Meier analysis showed that patients with CCA in the high 14-3-3? expression group had a worse outcome,and multivariate Cox regression analysis indicated its independent prognostic value.Moreover,scratch assay,Transwell assay,EMT marker detection assay and lung metastasis model in nude mice were employed and found that knocking down 14-3-3? expression could inhibit the invasive metastatic ability of Hu CCT1 cells,while upregulating 14-3-3? expression promoted the invasive metastatic ability of RBE cells.We found that inhibition of 14-3-3? inhibited the tumor stem cell properties of CCA cells by in vitro sphere-forming assay,flow cytometry,stemness marker detection assay and in vivo limiting dilution tumor-forming assay,while up-regulation of 14-3-3? expression inhibited the reverse.We treated CCA cells with gemcitabine,and 14-3-3? expression level was significantly increased.Moreover,CCK8 assay showed that the IC50 values of highly 14-3-3? expressed CCA cells were significantly higher.Conclusions 14-3-3? is highly expressed in CCA tissues,and is closely associated with lymph node metastasis,TNM stage and pathological differentiation.Moreover,14-3-3? is proved as an independent prognostic factor on the overall survival of CCA patients.And 14-3-3? is closely related to the ability of invasion and metastasis,property of cancer stem cells and chemotherapy resistance of CCA.Part ? Study on the role of 14-3-3? in regulating ferroptosis and its mechanismsObjective Cholangiocarcinoma(CCA)is a highly lethal disease with limited therapeutic options.Recent studies suggest that targeting ferroptosis in cancer cells may be a promising therapeutic approach.However,there are no studies exploring the role of ferroptosis in CCA.The aim of this study was to explore the role of 14-3-3? in regulating ferroptosis in cholangiocarcinoma and its mechanisms.Methods Ferroptosis inducer(Erastin)and ferroptosis inhibitors(ferrostatin-1 and liproxstatin-1)were used to verify whether Erastin induced ferroptosis in Hu CCT1,RBE and Gem-Hu CCT1.CCK8 assay,malondialdehyde(MDA)assay,Lipid ROS level assay,glutamate release assay,and glutathione content assay were employed to analyze the effects of 14-3-3? on Erastin-induced ferroptosis in CCA cells.Then,the regulatory mechanisms of 14-3-3? on Erastin-induced ferroptosis in CCA were explored by Western Blot,q RT-PCR and immunoprecipitation assay(Co-IP).Results CCK8 assay showed that Erastin inhibited the cell viability of Hu CCT1,RBE and Gem-Hu CCT1 in a dose-dependent manner,while ferrostatin-1 and liproxstatin-1 reversed these effects.Moreover,Gem-Hu CCT1 was more sensitive to the effect of Erastin.With the treatment of Erastin,the 14-3-3? expression was elevated in Hu CCT1 and RBE cells.Upregulation of 14-3-3? expression promoted Erastininduced ferroptosis in RBE cells and increased intracellular MDA production and Lipid ROS accumulation,whereas ferrostatin-1 and liproxstatin-1 inhibited the effects.Knockdown of 14-3-3? inhibited Erastin-induced ferroptosis in Hu CCT1 cells with decreased intracellular MDA production and Lipid ROS accumulation.In vivo experiments verified that upregulation of 14-3-3? promoted the antitumor effects of Erastin in nude mice.Further studies revealed that upregulation of 14-3-3? in RBE cells resulted in the inhibition of glutamate release and the decrease of intracellular GSH and increase of GSSG.In contrast,knockdown of 14-3-3? expression promoted the release of glutamate in Hu CCT1 cells with the increased intracellular GSH level was and decreased GSSG level.Upregulation of 14-3-3? in RBE cells decreased the expression level of SLC7A11 in protein and m RNA.Moreover,14-3-3? inhibited the Erastin-induced elevation of SLC7A11 protein expression.And knockdown of 14-3-3? promoted Erastin-induced elevation of SLC7A11 protein expression in Hu CCT1 cells.What's more,the half-life of GPX4 protein was detected using cycloheximide tracking assay.We found that upregulation of 14-3-3? significantly shortened the stabilization period of GPX4 protein in Hu CCT1 cells after Erastin treatment,while knockdown of 14-3-3? promoted the stability of GPX4 protein in RBE cells.CO-IP results showed that 14-3-3? and GPX4 proteins interacted with each other,and upregulation of 14-3-3? enhenced the interaction.Conclusions Erastin could induce ferroptosis in CCA cells,and the drug-resistant strains are more sensitive to ferroptosis inducer Erastin.14-3-3? promotes Erastin-induced ferroptosis in CCA cells.Moreover,14-3-3? inhibits system Xc-function and SLC7A11 expression.In addition,14-3-3? could interact with GPX4 protein,thereby inhibiting the degradation of GPX4 protein during Erastin-induced ferroptosis in CCA.