| Background:Esophageal carcinoma(ESCA)is one of the most common cancers in the world,which is mainly divided into esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC).The incidence and mortality of ESCA are at the forefront of all cancers.About 50 million people die from ESCA each year,accounting for 5.3%of all cancer deaths in the world,and showing significant geographical differences.China is the highest incidence and mortality of ESCA in the world,and North Sichuan and Yanting County are one of the high incidence areas of ESCA in China.The incidence of ESCA is high in the world and is increasing year by year.In this study,the expression and prognosis of YY1 in ESCA and its relationship with clinicopathological parameters were analyzed based on TCGA database.The possible molecular mechanism of YY1 in ESCA was discussed from gene mutation,immune cell infiltration and functional annotation analysis.Finally,the effects of YY1 on cell proliferation and glutamine metabolism of ESCC lines ECA109 and TE-1 were analyzed by cell experiments in vitro,and the potential molecular mechanism was preliminarily explored,which provides a theoretical basis for the development of new clinical treatment strategies for ESCC.Objective:1.Based on TCGA database,the expression and prognosis of YY1 in ESCA were analyzed,as well as its relationship with clinicopathological parameters of patients.To explore the correlation between YY1 and ESCA.2.Explore the expression of YY1 in ESCA,and analyze the correlation between YY1 and the patient’s pathological information3.YY1 was knocked out and overexpressed in ESCC to explore the possible role and molecular mechanism of YY1 in promoting the occurrence and development of ESCC.To provide a theoretical basis for the development of new clinical treatment strategies for ESCC.Methods:1.Using UCSC(www.genome.ucsc.edu),NCBI(https://www.ncbi.nl m.nih.gov)and SMART(http://smart.embl-heidelberg.de/)analyzed the c onserved functional domains of YY1 proteins in different species.2.UALCAN database was used to analyze the relationship between the expression of YY1 mRNA in ESCA tissues and its clinicopathological parameters.Kaplan-Meier plotter was used to analyze the prognostic value of YY1 mRNA expression in ESCA.CbioPortal website was used to explore,visualize and analyze the YY1 gene change analysis and TCGA data set in ESCA.3.TIMER was used to explore the correlation between the expression level of YY1 gene and the degree of immune cell infiltration,and the effect of YY1 gene somatic cell copy number change(SCNAs)on immune cell infiltration.GEPIA2 was used to analyze the relationship between the expression of YY1 in ESCA tumor,normal ESCA and GTEx and immune cell gene markers.TISIDB(http://cis.hku.hk/TISIDB/)was used to study the relationship between YY1 expression and immune subtypes and ESCA-related chemokines.4.LinkedOmics(http://www.linkedomics.org/)website)was used to analyze YY1-related expressed genes in TCGA-ESCA dataset.The biological functions of YY1-related expressed genes were classified by DAVID analysis tool(https://david.ncifcrf.gov/).Biological process(BP),molecular function(MF),cellular component(CC)and KEGG pathway were selected as analysis items.5.GSCALite(http://bioinfo.life.hust.edu.cn/web/GSCALite/)tumor Genome Analysis platform)was used to analyze the tumor pathway activity,GDSC and CTRP drug sensitivity of TCGA-ESCA data set.6.From July 2015 to March 2016,31 pairs of pathologically proved ESCC and paracancerous tissues were collected from the Department of Cardiothoracic surgery of the affiliated Hospital of North Sichuan Medical College.Immunohistochemical staining,Western Blot and RT-qPCR were used to detect the expression and localization of YY1 in ESCC and matched normal esophageal epithelial tissues.7.Normal esophageal epithelial cell lines HET-1A and ESCC cell lines ECA109 and TE-1 were cultured in vitro,and the expression of YY1 was quantitatively detected by RT-qPCR.8.The expression of YY1 was knocked down by siRNA at two sites(YY1 siRNA1,YY1 siRNA2),and a lentiviral-mediated YY1-overexpressed plasmid GV492-PCMV-PURO-YY1-OE(YY1-OE)infected with ESCC cell lines ECA109 and TE-1 was constructed.Western Blot and RT-qPCR were used to detect the inhibitory effect and overexpression of siRNA and shYY1 on the protein and mRNA expression of YY1 in ESCC cell lines ECA109 and TE-1.9.The effects of knockout and overexpression of YY1 on the proliferation,cloning and migration of EC A109 and TE-1 were detected by CCK-8 test,clone formation test and wound-healing assay.10.The expression of glutamine metabolism related proteins glutamine transporter(ASCT2),glutaminase(GLS)and glutamate dehydrogenase(GLUD1)in YY1 knock-down and overexpression cell lines were detected by Western Blot and RT-qPCR.11.The metabolites were detected by glutamine test box and glutamate test box to analyze the effects of YY1 knock-down and overexpression on the contents of glutamine and glutamate in ECA109 and TE-1,and to clarify the regulatory effect of YY1 on glutamine uptake and decomposition.Results:1.The YY1 genes of patients with ESCA were systematically analyzed by using a variety of bioinformatics databases.The results showed that the expression of YY1 mRNA in ESCA tissues was significantly higher than that in normal tissues(P<0.01).In addition,we also studied the relationship between clinicopathological data and YY1 expression in patients with ESCA.The expression of YY1 mRNA in ESCA was significantly correlated with all clinicopathological parameters.2.In this study,we found that YY1 was significantly correlated with the abundance of immune cells,immune gene markers and chemokines.YY1 is mainly involved in the activation of apoptosis pathway,cell cycle and DNA damage response pathway.In addition,YY1 with low expression was resistant to 23 and 55 drugs in GDSC and CTRP,respectively.3.The expression of YY1 in cancer tissues and adjacent noncancerous tissues of ESCC patients was evaluated by immunohistochemical staining,Western Blot,and RT-qPCR in 31 pairs,and it was found that the expression of YY1 in ESCC tissues was significantly higher than that in normal adjacent tissues(P<0.05).4.By detecting the expression of YY1 in ESCC cell lines TE-1,ECA 109 and KYSE150 and normal esophageal epithelial cell line HET-1A,it was found that the expression level of YY1 in TE-1 and EC A109 was significantly higher than that in normal esophageal epithelial cell line HET-1A(P<0.05).It can be used as experimental subjects to further study the cellular biological function.5.After YY1 gene knockout,it was found that the proliferation,clone formation and migration of ESCC cell line TE-1,ECA109 were significantly inhibited(P<0.05),while the proliferation and migration of two ESCC cell lines were significantly promoted after overexpression of YY1 gene(P<0.05).6.In this study,we found that glutamine metabolism may be involved in the process of ESCA by David method.At the same time,it was found that glutamine uptake and glutamate production decreased significantly in ESCC cell lines down-regulated by YY1(P<0.05),while glutamine uptake and glutamate production increased significantly after overexpression of YY1(P<0.05).7.In this study,it was found that the protein expression levels of glutamine metabolism related proteins ASCT2,GLS and GLUD1 were significantly down-regulated after YY1 knockdown,while the protein expression levels of ASCT2,GLS and GLUD1 were significantly up-regulated after YY1 overexpression.Conclusion:We found that the abnormally high expression of YY1 in ESCC may be a potential marker of ESCC and participate in many biological processes such as ESCC proliferation,clone formation and migration.YY1 may affect glutamine metabolism by regulating the expression of glutamine metabolism related genes ASCT2,GLS and GLUD1,and then promote tumor cell proliferation,clone formation and migration.Therefore,transcription factor YY1 is expected to become a diagnostic marker and therapeutic target of ESCC. |
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