Minichromosome Maintenance 6 (MCM6) Identified By Bioinformatics Analysis And Experimental Validation In Esophageal Squamous Cell Carcinoma

Background:Esophageal squamous cell carcinoma(ESCC),the main subtype of esophageal cancer(EC),is a common lethal type of cancer with a high mortality rate.The present study aimed to select key relevant genes and identify potential mechanism involved in the development of ESCC based on bioinformatics analysis.Methods:1.Four RNA-seq datasets(GSE63941,GSE26886,GSE17351 and GSE77861)related to ESCC were collected from the GEO database and analyzed through the online analysis tool GEO2R.The differentially expressed genes(DEGs)in each dataset were obtained.The selection criteria are as follows:adj P<0.05,|logFC|?1.The intersection of the DEGs were obtained using a Venn analysis diagram and used for the subsequent analysis.2.Then the protein-protein interaction(PPI)were constructed by STRING analysis,and the modules were verified by Cytoscape,in which the key genes have a high connectivity degree with other genes.Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway were subsequently filtered out to analyze the development of ESCC by online analysis tools DAVID.The MCM6 was selected for further verification.3.The expression levels of MCM6 were preliminarily assessed using the ONCOMINE,GEPIA and UALCAN databases in EC and other cancers.4.Tissue specimens and adjacent control tissues and pathological data,confirmed by histology as ESCC,were collected.The expression of MCM6 protein were detected by immunohistochemistry,and the expression of MCM6 mRNA in ESCC cell lines,EC 109 and KYSE30,were detected by RT-qPCR.5.By Knocking down the expression of MCM6 by siRNAs,Cell counting kit-8(CCK-8),Transwell assays,wound healing and flow cytometry were used to determine the proliferation,migration and invasion,apoptosis,cell cycle of ESCC cells.Results:1.4 eligible data sets(GSE63941,GSE26886,GSE17351 and GSE77861)were found through GEO database,and 68 genes(48 up-regulated genes,20 downregulated genes)were got by Venn interactive analysis.2.A total of 24 core DEGs were finally obtained through protein interaction analysis.GO annotation and KEGG pathway analysis by DAVID online analysis tools found that these core genes are mainly related to DNA replication and cell cycle.The gene MCM6 that is obviously enriched and analyzed was selected for subsequent experimental verification.3.MCM6 is highly expressed in ONCOMINE,GEPIA,and UALCAN,and it is related to tumor grade,race and other factors.The expression of MCM6 is also elevated in esophageal adenocarcinoma,Barrett's esophagus and most tumor tissues.4.Experimental validation revealed that MCM6 expression was significantly elevated in ESCC tissues(76.5%)than control tissues(26.7%)by Immunohistochemical analysis.MCM6 was elevated in EC 109 and KYSE30 cell lines by RT-qPCR than normal cell line Het-1.The results were consistent with those of bioinformatics analysis.5.Furthermore,the knockdown of MCM6 by siRNAs inhibited cell proliferation,invasion and migration,promoted cell apoptosis,and significantly blocked cell cycle S/G2 transformation.Conclusions:1.24differentially expressed genes related to ESCC were explored in GEO database,and all genes play key roles in the occurrence and development of ESCC.2.The expression of MCM6 gene is significantly up-regulated in EC tissues and cell lines.3.MCM6 is related to cell proliferation,migration,invasion and apoptosis.