BRD4 And Mitochondrial Autophagy-related Proteins As Prognostic Markers And Mechanisms Of Esophageal Cancer

Esophageal cancer(EC),a common digestive tract tumor,ranks eighth among all cancers worldwide.According to histological classification,esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC)are the two main pathological types of esophageal cancer,of which the former accounts for 90%of esophageal cancer cases in China.Surgery is the most common treatment for ESCC,as well as postoperative adjuvant chemoradiotherapy.Although conventional care significantly improves median survival in patients with ESCC,the recurrence rate of ESCC remains high.In recent years,patients with ESCC have had a low 5-year overall survival rate due to local invasion and distant metastasis.BRD4(Bromodomain Containing 4)is a member of the BET family(Bromodomain and Extra-terminal Tomain)and may be a potential target for cancer therapy due to its important role in transcriptional and epigenetic regulators of the cell cycle.In addition,BRD4 interacts with many well-known signaling pathways such as NF-κB(Nuclear factor-kappa B),AKT(Protein kinase B),etc.,and plays a cancer-promoting role in some tumors such as lung cancer,breast cancer,gallbladder cancer,etc.However,the molecular mechanism and therapeutic potential of BRD4 in esophageal squamous cell carcinoma are unclear.Mitochondrial autophagy is closely related to the occurrence and development of cancer,and in malignant tumors such as esophageal cancer,mitochondrial autophagy is closely related to the abnormal activation and proliferation of tumor cells,which has both cancer-promoting and cancer-suppressing effects.Therefore,the search for mitochondrial autophagy genes associated with esophageal cancer may provide new ideas for the treatment of esophageal cancer patients.It is of great significance to analyze the prognosis of mitochondrial autophagy-related genes in different subtypes of esophageal cancer,construct prognostic models,and look for immune cell infiltration with significant differences between subtypes.This project is divided into two parts,respectively,to study BRD4 and mitophagy-related protein as prognostic markers and mechanisms of esophageal cancer.This paper aims to explore the mechanism of action of BRD4 as a possible biomarker of ESCC participation in ESCC,and to find mitochondrial autophagy-related genes related to the prognosis of esophageal cancer,so as to provide new predictors and therapeutic targets for the treatment of esophageal cancer patients.Part Ⅰ BRD4 promotes ESCC progression via regulating the AKT/NF-κB signaling pathwayObjective1.To explore the molecular mechanism and therapeutic potential of BRD4 in ESCC;2.To determine whether BRD4 can enhance the migration and proliferation ability of esophageal squamous cells by activating the AKT/NF-κB pathway?MethodsFresh tumor tissue and adjacent normal tissue samples from 8 patients with esophageal squamous cell carcinoma were collected from Qilu Hospital of Shandong University.The expression of BRD4 in esophageal squamous cell carcinoma and normal tissues(P<0.05)was verified by the UALCAN database of the cancer genome atlas(TCGA).The western blotting method was used to detect BRD4 expression in 8 cases of tumor fresh tissue and adjacent normal tissue.The relationship between expression levels and clinicopathological features of ESCC patients in the TCGA database was evaluated by logistic regression analysis.Kaplan-Meier survival analysis was used to study the relationship between BRD4 expression levels and survival prognosis.In vitro experiments,BRD4 protein was transfected into human ESCC cell line Eca109 and its expression was knocked down,the effect of BRD4 on the migration ability of esophageal squamous cell carcinoma cells was verified by Transwell migration experiment and scratch experiment,and the effect of BRD4 on the proliferation ability of esophageal squamous cell carcinoma cells was verified by CCK8 experiment and cloning formation experiment.Gene set enrichment analysis(GSEA)studies the signaling pathway associated with BRD4 high expression in ESCC,and Western blotting analyzes the relationship between BRD4 and AKT/NF-κB pathway,and verifies whether BRD4 is positively correlated with other molecules in the pathway through co-expression analysis.and exploring the mechanism of action of BRD4 in esophageal squamous cell carcinoma.All statistical analyses were performed using GraphPad Prism 7(GraphPad Software,Inc.).Paired student’s t test was used to compare differences in BRD4 protein levels between paracancerous and paracancerous tissues.Data are expressed as mean±standard deviation.Individual experiments were repeated 3 times.P<0.05 was a significant difference between groups.Results1.The average expression level of BRD4 in ESCC tissues and ESCC cell lines is elevated compared to normal esophageal tissue and esophageal cells;2.BRD4 expression level was positively correlated with the adverse prognosis of clinical T stage of ESCC.ESCC patients with high BRD4 expression had a lower survival rate than ESCC patients with low BRD4 expression(P<0.05).3.Western blotting quantified the expression levels of BRD4 in different esophageal squamous cell cell lines(Eca140,Eca109,Eca150),and the BRD4 expression levels were inconsistent between cell lines,which were highly expressed in the Eca109 cell line;4.Effect of downregulation of BRD4 on cell migration and proliferation in esophageal squamous cell carcinoma cells:(1)Transfection was evaluated using qRT-PCR and western blotting,and transfection of siRNA-BRD4 reduced BRD4 expression by about 4-fold compared to control cells(P<0.01).(2)Transwell and scratch experiments showed that downregulation of BRD4 inhibited the migration of Eca109 cells(P<0.0001).(3)CCK8 proliferation experiments and cloning formation experiments showed that siRNA inhibited BRD4 expression and significantly inhibited the proliferation of Eca109 cells(P<0.05)and reduced the number of colonies formed.5.BRD4 targets the AKT/NF-κB signaling pathwayGSEA enrichment analysis found that genes regulating AKT and NF-κB signaling pathways were significantly enriched in BRD4 high-expression samples,and co-expression network analysis also showed that BRD4 and AKT were positively correlated with many proteins in NF-κB signaling pathway(P<0.05).Western blotting measured the expression levels of AKT,NF-κB and their phosphorylated forms,and found that the downregulation of BRD4 significantly inhibited the phosphorylation of AKT and NF-κB in ESCC cells.ConclusionBRD4 is highly expressed in esophageal squamous cell carcinoma,which is not conducive to the survival prognosis of esophageal squamous cell carcinoma patients.BRD4 may enhance the migration and proliferation ability of esophageal squamous cells by activating the AKT/NF-κB pathway,and accelerate tumor development.BRD4 may become a biomarker and therapeutic target for patients with esophageal squamous cell carcinoma,and has potential value in prognosis and treatment.Part Ⅱ Mitophagy-related genes predict EC prognosis and immune responseObjective1.Analyze mitochondrial autophagy genes related to prognosis in different subtypes of esophageal cancer and construct prognostic models;2.Analysis of the composition of immune cells between subtypes and the level of infiltration.MethodsWe downloaded 185 esophageal cancer samples from the TCGA database,including gene expression level data and clinical follow-up data,and 179 esophageal cancer samples from the GEO database for subsequent validation analysis.Obtaining the best mitochondrial autophagy subtype by ConsensusClusterPlus analysis method;The Kaplan-Meier curve method was used to evaluate the correlation of survival prognosis between different subtype groups.Chi-square test analyzes the correlation between subtypes and clinical information;Differential genetic analysis between different subtypes using LIMMA packs(threshold setting:ADJ.P.Value<0.05&|log2FC|>1);Univariate Cox regression analysis found genes with significant prognosis,and the LASSO algorithm screened key genes.Stepwise Cox regression analysis constructs risk scores and divides high and low risk groups.The Kaplan-Meier curve method assesses the association between high-and low-risk groups and actual survival prognostic information.Independent prognostic factors were screened by univariate and multivariate COX analysis to construct nomograms.In addition,we used the CIBERSORT method to calculate the composition ratio of 22 immune cell,and the matrix fraction and immune score of tumor samples were calculated by the ESTIMATE algorithm.At the same time,we used the Wilcoxon test to compare the expression differences between the immune checkpoint gene and the HLA family gene between the high-and low-risk groups,and the difference in IC50 between the high-and low-risk groups of 138 chemotherapy drugs.The relationship between mitochondrial autophagy subtypes and high-and low-risk groups was analyzed using the ggalluvial package in R3.6.1.ResultsWe identified 7 mitochondrial autophagy genes associated with esophageal cancer prognosis(including PTPN4,ALKBH4,IL6,FN3KRP,HSDL1,B3GNT2,CCT4);There was a significant correlation between high-and low-risk groups and actual prognosis;Prognostic factors Stage and RiskGroup constructed Nomogram,which correlated significantly with patient outcomes.Eleven significantly different immune cells in the two subtypes were obtained,and 10 significantly different immune checkpoint genes were obtained.ConclusionSeven mitochondrial autophagy genes associated with the prognosis of esophageal cancer may become key prognostic genes and novel therapeutic targets for esophageal cancer.