The Role Of Long Non-coding RNA H19 Regulated By Epstein-Barr Virus In Gastric Carcinoma Development

Objective:(1)To screen differentially expressed long non-coding RNAs(lnc RNAs)of AGS cell line before and after infected by Epstein-Barr virus,trying to provide theoretical and experimental basis to clarify the tumorigenic mechanism of EBV.(2)To investigate expression levels of the long non-coding RNA H19 in EBV-positive and-negative gastric cancer cell lines and tissue samples,compare the difference of lnc RNA H19 expression levels between EBV-positive and-negative samples and explore the impact of EBV infection on H19 promoter methylation levels and H19 imprinting status in gastric cancer.Methods:(1)High throughout transcriptome sequencing(RNA-Seq)was adopted to analyze the transcriptome of AGS cells before and after EBV infection.(2)The obtained sequencing data was analysed by using bioinformatic technology including expression levels of lnc RNAs analysis,target gene prediction of differentially expressed lnc RNAs,and the enrichment analysis of Go(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)for the putative target genes.(3)Expression levels of lnc RNA H19 in EBV-positive(GT-38,GT-39,SNU719,AGS-EBV)and-negative(MKN-45,SGC7901,HGC-27,AGS)gastric cancer cell lines,EBV-associated gastric cancinoma(EBVa GC)and EBV-negative gastric canccinoma tissue samples were detected by real time fluorescent quantitative RT-PCR(q RT-PCR).(4)Bisulfate Genomic Sequencing(BGS)and Sequenom Mass Array Flight Mass Spectrometry were used to analyze H19 promoter methylation status in EBV-positive and-negative gastric cancer cell lines,EBVa GC and EBVn GC tissue samples;According to the 434G/T polymorphism site in the detected sequence region,the allele-specific methylation status of H19 promoters in 434G/T heterozygous gastric cancer tissues samples were analyzed.(5)Based on the existence of Rsa I restrict enzyme site in the 5th exon of H19,imprinting status of H19 was detected by using restrictive fragment length polymorphisms(RFLP)and PCR(RFLP-PCR)in EBVa GC and EBVn GC tissue samples.Results:(1)Compared with AGS cells,879 lnc RNAs were differentially expressed in AGS-EBV,including 47 up-regulated and 832 down-regulated.Among them,24 lnc RNAs were not expressed in AGS,while 243 were not detected in AGS-EBV.Some differentially expressed lnc RNAs such as H19 and CCAT1 have been reported to be associated with development of distinct neoplasms.(2)Target genes of some differentially expressed lnc RNAs were successfully predicted.By using enrichment analysis of GO term,we found that these target genes were involved in various biological processes,cellular components and molecular functions.Some target genes participated in apoptotic process,cell cycle arrest and cell proliferation.Enrichment analysis of KEGG showed that the target genes might participate in pathways including oxidative phosphorylation,metabolic pathways,endocytosis,salivery secretion,pancreatic secretion and some metabolic disease such as Alzheimer’s disease,Huntington’s disease and Parkinson’s disease.(3)lnc RNA H19 expression level in EBV-positive cell lines was significantly lower than that in EBV-negative ones(0.04789±0.02991 vs 4.061±0.4192,P<0.001);Obviously lower lnc RNA H19 expression level was also observed in EBVa GC than in EBVn GC tissue samples EBVn GC(0.7024±0.4000 vs 3.646±0.7058,P<0.001).(4)H19 promoters in EBV-positive cell lines all showed hypermethylated status,while in EBV-negative ones only a few Cp G sites were methylated.Methylated ratio of H19 promoter Cp G sites was significantly higher in EBV-positive cell lines than that in EBV-negative ones.Results from BGS and Sequenom Mass Array Flight Mass Spectrometry showed that mean methylated ratios of H19 promoters were nearly fifty percent both in EBVa GC and EBVn GC tissues.There was no significant difference between the two groups(P=0.779,P=0.729).The distribution of hypermethylated and hypomethylated samples in the two groups was similar(P=0.721).Allele-specific methylation status analysis of H19 promoter in 434G/T heterozygous gastric cancer tissues samples demonstrated that in 5 EBVa GC tissues,one H19 allele was almost completely methylated and the other one was somewhat methylated.H19 alleles in Sample PL455 were both hypermethylated.However,in 5 EBVn GC tissues,one H19 allele was almost completely methylated and the other one was almost completely unmethylated.H19 alleles in Sample Q589 were both almost unmethylated.It seemed that higher methylated H19 promoters existed in EBVa GC than in EBVn GC.(5)In 39 EBVa GC samples and 86 EBVn GC samples,24 and 28 heterozygous samples at Rsa I restrict enzyme site were obtained.6(25%,6/24)in EBVa GC and 6 in EBVn GC(21.43%,6/28)showed H19 biallelic expression.Frequency of H19 loss of imprinting was not significantly different between the two groups(P=1.00).Conclusions:(1)Stable infection of EBV can lead to changes in expression levels of lnc RNAs.Downregulated lnc RNAs were more than upregulated ones.Some differentially expressed lnc RNAs were associated with development of tumors.(2)The differentially expressed lnc RNAs were involved in distinct cellular biological activities,which suggested that EBV may have impacts on protein-encoding genes through lnc RNAs,therefore affect transcription regulation more extensively and be involved in development or progression of EBV-associated tumors.(3)EBV infection impacted greatly on lnc RNA H19 expression in gastric cancer cells.EBV might be involved in the development and progression of EBV-associated gastric cancer by regulation of lnc RNA H19.(4)Methylated level of H19 promoter in EBV-positive cell lines was higher in EBV-negative ones.Lnc RNA H19 expression level was tightly associated with methylated level,which was an important factor of H19 expression regulation.However,in tissue level,methylated level of H19 promoter between EBV-positive and –negative groups was not significantly different.This suggested that other regulation factors may also play roles.(5)There were H19 loss of imprinting in some gastric cancer tissues,which might be one of the causes leading to high H19 expression level in gastric cancer.EBV infection had no impact on H19 loss of imprinting in gastric cancer.