Screening And Related Functional Study Of Differentially Expression Key Genes In Esophageal Cancer

China is a country with a high incidence of esophageal cancer,accounting for more than half of the global morbidity and mortality.Due to the difficulty of early diagnosis,most patients with esophageal squamous cell carcinoma(ESCC)are already in the middle and late stages of the disease when they are diagnosed,which is also the main reason for the low five-year survival rate of esophageal cancer in China.In order to improve the survival of patients with esophageal cancer,it is urgent to find biomarkers and therapeutic targets for diagnosis and prognosis.A large number of studies have shown that the occurrence and development of esophageal cancer is a process interacted by multiple stages、genes,and factors,however,the exact pathogenesis is not clear.There have been a large number of reports on the application of gene expression profiling and high-throughput transcriptome technology to analyze esophageal cancer-related genes.This provides a good research direction for the discovery of molecular markers related to esophageal cancer diagnosis.Epigenetic modifications,such as DNA methylation,have been extensively studied and used in the diagnosis and treatment of cancer.Abnormal hypermethylation is associated with the inactivation of tumor-associated genes in a variety of tumor types.Therefore,analyzing the correlation between gene transcription sequencing data and DNA methylation modification data is of great significance for clarifying the epigenetic regulation mechanism of esophageal cancer.In this study,m RNA and DNA methylation data were downloaded from the Cancer Genome Atlas(TCGA)database to screen and analyze differentially expressed genes and differentially methylated genes in esophageal cancer tissues and adjacent tissues.Then the differential expression genes and differential methylation genes were verified and the key genes were screened.The expression of the key genes in esophageal cancer was further analyzed,and the relationship between the expression of the key genes and clinicopathological data of esophageal cancer was discussed.Finally,the effects of the selected key genes on the proliferation,migration,invasion and apoptosis of esophageal cancer cell lines were observed by knocking down and overexpressing them.This study will contribute to the diagnosis of esophageal cancer,the development of biomarkers and targeted therapy drug research and development to provide a theoretical basis.Part One Screening and Analysis of Differentially Expressed Genes and Methylation Genes in Esophageal CancerObjective:To screen differentially expressed genes and differentially methylated genes in esophageal cancer tissues and adjacent tissues in order to obtain new targets for diagnosis and treatment.Methods:1.The m RNA and DNA methylation data of esophageal cancer tissues and adjacent tissues were downloaded from TCGA database,and the differentially expressed genes and differentially methylated genes were screened by R language software.2.Weighted gene co-expression network analysis(WGCNA)was used to analyze the downloaded m RNA data to determine the best module,and KEGG enrichment analysis and GO analysis were used to study the biological function of the genes in the best module.3.The key genes were identified by association analysis of differentially expressed genes and differentially methylated genes.Results:1.A total of 2408 differentially expressed genes were screened,of which1311 were up-regulated and 1097 down-regulated.2.A total of 49 modules were identified by co-expression network analysis of the downloaded m RNA data by WGCNA,of which the royal blue module was identified as the best module.KEGG enrichment analysis showed that gastric acid secretion and protein digestion and absorption were the only two significantly enriched signaling pathways.GO-enrichment analysis showed that ion transport and transmembrane transport were the biological processes of significant enrichment,components of cell membrane and cell membrane were the cellular components of significant enrichment,and voltage-gated ion channel activity and chloride channel activity were the molecular functions of significant enrichment.3.A total of 5134 differentially methylated sites were screened from TCGA database,including 4151 hypermethylated sites and 983hypomethylated sites.There were 1335 differentially methylated genes,including 1098 hypermethylated genes and 237 hypomethylated genes.4.The core genes ATP4A,ATP4B,CCKAR,MFSD4 and ESRRG in the sapphire blue module were found to be both highly methylated and lowly expressed genes by association analysis of differentially expressed genes and differentially methylated genes.In addition,the ESRRG gene was found to be most closely related to the sapphire blue module among the five genes,suggesting that ESRRG may play a more important role in the development of esophageal cancer.Summary:A total of 2408 differentially expressed genes and 5134differentially methylated sites were obtained by screening.In the WGCNA analysis,the royal blue module was considered the best module.The core genes ATP4A,ATP4B,CCKAR,MFSD4 and ESRRG in the sapphire blue module are both hypermethylated and low expressed genes,indicating that the above five genes may play an important role in the occurrence and development of esophageal cancer.In addition,ESRRG gene was found to be most closely related to the sapphire blue module among the five genes,suggesting that ESRRG may play a more important role in the occurrence and development of esophageal cancer.Part Two Verification and Further Analysis of Differentially Expressed and Differentially Methylated Genes in Esophageal Cancer.Objective:To identify the differentially expressed genes and methylated genes,and to find the key genes which may play an important role in the development of esophageal cancer.Methods:1.The selected ATP4A,ATP4B,CCKAR,MFSD4 and ESRRG genes were verified by GEO database.2.ATP4A,ATP4B,CCKAR,MFSD4 and ESRRG genes were analyzed by TCGA database.3.The ATP4A,ATP4B,CCKAR,MFSD4 and ESRRG genes in esophageal cancer tissues and paracancerous tissues were verified in vitro by reverse transcription quantitative PCR(qRT-PCR).4.Through the association analysis of the first part,the key target genes which play an important role in the occurrence and development of esophageal cancer were identified.Results:1.The results of GEO database analysis showed that the expression levels of ATP4A,ATP4B,MFSD4 and ESRRG genes were all down-regulated in esophageal cancer tissues except CCKAR,and the down-regulation level of ESRRG gene was the most obvious(P=3.2e-05).The results showed that ESRRG may play a more important role in the occurrence and development of esophageal cancer.2.The area under curve(AUC)of ATP4A,ATP4B,CCKAR,MFSD4 and ESRRG were 0.605,0.645,0.805,0.761 and 0.747,respectively.These results suggest that CCKAR,MFSD4 and ESRRG genes may be potential biomarkers for the diagnosis of esophageal cancer.1.3.he results of qRT-PCR showed that the expressions of ATP4A,ATP4B,CCKAR,MFSD4 and ESRRG in esophageal cancer tissues were significantly lower than those in paracancerous tissues(all P<0.001),and ESRRG was the most significantly differentially expressed.These results are consistent with the results of bioinformatics analysis.4.Through the above verification and the association analysis with the first part,we found that ESRRG gene may play an important role in the occurrence and development of esophageal cancer.Summary:The results of screening differentially expressed genes in esophageal cancer by multiple bioinformatics methods are reliable.The most significant down-regulation of ESRRG gene expression was observed in esophageal cancer tissues by both GEO database and qRT-PCR in vitro.Diagnostic analysis showed that CCKAR,MFSD4 and ESRRG genes may be potential biomarkers for the diagnosis of esophageal cancer.Through the comprehensive analysis of the first part,we found that ESRRG gene may play an important role in the occurrence and development of esophageal cancer,and can be used as a new target for potential diagnosis and treatment of esophageal cancer.Part Three The Expression of ESRRG in Esophageal Cancer and its Clinical Correlation StudyObjective:To investigate the expression level of ESRRG in esophageal carcinoma tissues and paracancerous tissues,and analyze the relationship between the expression level of ESRRG and clinicopathological features.Methods:1.Clinical specimens of patients with esophageal cancer were collected and patient information was collected.2.The expression of ESRRG was detected by qRT-PCR in esophageal cancer tissues and adjacent tissues.3.The relationship between the expression of ESRRG and the clinicopathological characteristics was analyzed in patients with esophageal cancer.4.The expression of ESRRG was detected by immunohistochemistry in esophageal cancer and paracancer tissues.Results:1.The expression of ESRRG in esophageal carcinoma was significantly lower than that in paracancerous tissues(P<0.0001).2.The expression of ESRRG was not correlated with sex,age,smoking,drinking and tumor location(all P>0.05),but closely correlated with lymph node metastasis and TNM stage(all P<0.05).Patients with low ESRRG expression were more likely to have lymph node metastasis(χ~2=4.571,P<0.05),and TNM staging was later(χ~2=6.149,P<0.05).3.Immunohistochemical results showed that the expression of ESRRG in esophageal carcinoma was significantly lower than that in paracancerous tissues(P<0.0001).Summary:qRT-PCR showed that the expression of ESRRG in esophageal carcinoma was significantly lower than that in paracancerous tissues.The expression of ESRRG was not correlated with sex,age,smoking,drinking and tumor location,but closely correlated with lymph node metastasis and TNM stage.Immunohistochemistry showed that the expression of ESRRG in esophageal carcinoma was significantly lower than that in paracancerous tissues.These results suggest that ESRRG is negatively correlated with the occurrence and development of esophageal cancer.Part Four Effects of ESRRG on Proliferation,Migration,Invasion and Apoptosis of Esophageal Cancer Cell LinesObjective:To investigate the effects of knockdown and overexpression of ESRRG gene on proliferation,migration,invasion and apoptosis of esophageal cancer cell lines.Methods:ASO-NC and ASO-ESRRG were transfected into KYSE510cell line to knock down ESRRG;TE-1 cells were transfected with pc DNA3.1and pc DNA3.1/ESRRG to overexpress ESRRG.Then the expression of ESRRG m RNA in each group was detected.The effects of ESRRG on the proliferation,migration,invasion and apoptosis of esophageal cancer cell lines were detected by MTT,cell wound healing,Transwell and flow cytometry.Results:1.The results of qRT-PCR showed that the expression of ESRRG m RNA in ASO-ESRRG knockdown group was significantly lower than that in ASO-NC group(P<0.001).In TE-1 cell line,the expression of ESRRG m RNA in pc DNA3.1/ESRRG group was significantly higher than that in pc DNA3.1group(P<0.001).2.MTT showed that the proliferation ability of ASO-ESRRG group was stronger than that of ASO-NC group at 24h,48h and 72h in KYSE510 cell line(P<0.05).In TE-1 cell line,the proliferation of pc DNA3.1/ESRRG group was significantly lower than that of pc DNA3.1 group at 24h,48h and 72h(P<0.05).3.Cell scratch test showed that in KYSE510 cell line,the migration distance of ASO-ESRRG group with ESRRG knockout was significantly higher than that of ASO-NC group,and there was a significant difference between the two groups(P<0.01).In TE-1 cell line,the migration distance of overexpressing ESRRG in pc DNA3.1/ESRRG group was significantly lower than that in pc DNA3.1 group(P<0.01).4.Transwell cell invasion assay showed that the number of invasive cells in ASO-ESRRG group with ESRRG knockdown was significantly higher than that in ASO-NC group(P<0.01).In TE-1 cell line,the number of invasive cells in pc DNA3.1/ESRRG group was significantly lower than that in pc DNA3.1 group(P<0.01).5.Flow cytometry showed that in KYSE510 cell line,the apoptosis rate of ASO-ESRRG group was significantly lower than that of ASO-NC group(P<0.05).In TE-1 cell line,the apoptosis rate of pc DNA3.1/ESRRG group was significantly higher than that of pc DNA3.1 group(P<0.05).Summary:Knockdown ESRRG in esophageal cancer KYSE510 cell line,KYSE510 cell proliferation ability was significantly enhanced,migration and invasion ability was significantly enhanced,apoptosis rate was significantly reduced.Overexpression of ESRRG and TE-1 in esophageal cancer cell line TE-1 cell proliferation ability was significantly reduced,migration and invasion ability was significantly reduced,the proportion of apoptosis was significantly increased.These results suggest that ESRRG can inhibit the proliferation,migration and invasion of esophageal cancer cells,and promote their apoptosis.ESRRG gene is expected to become a new target for the treatment of esophageal cancer.