The Epigenetic Regulation Study Of Epstein-Barr Virus In Nasopharyngeal Carcinoma Cells

Research Background and Purpose:Nasopharyngeal carcinoma is a head and neck cancer that occurs in the south of China and seriously threatens the health of people in the southeast coast.Nasopharyngeal carcinoma is a polygenic genetic disease.Environmental factors also have an important impact on it.At the same time,the occurrence and development of nasopharyngeal carcinoma has complex and diverse links with EBV,but its pathogenesis is still unclear.Studies have shown that EBV plays a role in many tumors.EBV can induce the activation of some transcriptional regulatory elements in B-cell lymphoma,and participate in a series of complex biological regulation processes,which eventually raises the abnormal expression of some oncogenes,making resting B cells immortalized and cancerous.The gene regulation function of EBV may be its carcinogenic mechanism.Based on the gene regulation function of EBV,this study used the nasopharyngeal carcinoma cell line HONE1,which is stably expressed in the genome of nasopharyngeal carcinoma epithelial cells,to explore the relationship between the expression of genes associated with nasopharyngeal carcinoma and EBV,and to analyze the relationship between complex cellular regulation mechanisms of EBV and the development of nasopharyngeal carcinoma.By comparing with EBV negative and positive HONE1 cell line,we aimed to study the gene expression regulation and epigenetic functions of EBV,Inferingand verifying the gene regulation function of EBV in nasopharyngeal carcinoma,and thus to explore The carcinogenic mechanism of EBV in nasopharyngeal carcinoma.Methods1.High-throughput RNA deep sequencing(RNA-seq)method was used to study the difference of gene expression in EBV-positive and non-EBV-infected HONE1 cells,to analyze and identify the effect of EBV on cell gene expression in HONE1.2.The RT-qPCR method was used to verify the mRNA expression level of EBV positive and negative Honel cells,and the effect of EBV on SYK gene expression was studied.3.Chromatin immunoprecipitation and its deep DNA sequencing technology(ChIP-seq)were used to detect the changes of histone modifications such as H3K4mel,H3K4me3 and H3K27ac in the EBV positive and negative HONE1 cells,to explore the epgenetic function of EBV in nasopharyngeal carcinoma.4.Based on the analysis results of RNA-seq and ChIP-seq,the SYK locus with significantly increased expression and significant ChIP-seq signal change in EBV-positive HONE1 cells was identified,and the circular chromatin conformation capture coupled withnext-generation sequencing(4C-seq)technology was usedto detect the chromosomal spatial interaction of EBV in the genome,thereby verifying the gene regulatory mechanism of EBV gene in nasopharyngeal carcinoma.Results1.RNA-seq results told us that EBV caused up-regulation of 261 genes,down-regulation of 720 genes in Honel cells,and the 261 genes was create gene sets separately.The secondary classification of GOanalysis showed that the major cellular and biological regulation processes of EBV-induced upregulation genesof HONE1.The main cellular and biological regulation processes of HONE 1 up-regulated genes mainly constitute cells and cell components,mainly performing molecular functions of molecular binding and biochemical activity;COG classification shows that these up-regulated genes are mainly concentrated in lipid transport and metabolism,and overall functions,signal transduction,cell composition,etc.;GO enrichment classification showed that these up-regulated genes were significantly enriched in biological processes such as cholesterol,secondary alcohol biosynthesis,sterol biosynthesis and metabolic regulation,andnegative regulation of sodium ion transport.The KEGG enrichment classification revealed that these up-regulated genes had the highest number of genes enriched in the sterol and glycolipid biosynthetic pathways,and with the most significant difference;the RNA-seq data in IgV showed that the SYK gene was significantly expressed in the presence of EBV.2.RT-qPCR result verifies that the mRNA expression level of SYK gene was significantly up-regulated under the existence of EBV in HONE1 cells.3.ChIP-seq results showed that the SYK gene H3K4me3 ChIP-seq signal was significantly enhanced in EBV-positive Honel cells,indicating that the transcription level of SYK gene was enhanced in the presence of EBV;SYK gene was also observed in EBV-positive HONE1 cells.The H3K27ac ChIP-seq signals in the downstream region was significantly enhanced,and we speculated that there were enhancers and super enhancers that regulate the transcriptional function of the SYK gene promoter.4.The 4C results showed that there were enhancers downstream of the SYK gene,which regulated the SYK promoter region through chromatin spatial interaction,activated SYK transcriptional regulation,and up-regulated SYK gene expression.