Construction And Preliminary Validation The Regulatory Network Representing Interaction Between Epstein-Barr Virus-encoded MicroRNAs And Their Potential Target Host Genes In Nasopharyngeal Carcinoma

Epstein-Barr virus (EBV) is related to a broad spectrum of benign and malignant disease, including nasopharyngeal carcinoma (NPC). However, the exact mechanism by which EBV promotes oncogenesis is still unclear. Recently, a role in EBV-mediated transformation has been proposed for a newly described class of small non-coding RNAs called EBV miRNAs, which is encoded by EBV. EBV miRNAs are a new class of tumorigenic molecules, since2004found that the first EBV miRNA, we have just found a total of44EBV-encoded miRNAs, which can be divided into two groups:BART and BHRF1. But EBV miRNAs research is not very thorough in NPC, and most of44mature EBV miRNAs have no function studies reported.To determine how EBV-encoded miRNAs control the expression of host genes and to understand the potential role of viral miRNAs in NPC tumorigenesis, we profiled the expression of all44mature miRNAs encoded by EBV and the genome-wide human host cellular genes in NPC biopsies and nontumor nasopharyngeal epithelial tissues; we then performed an integrated and comprehensive analysis of EBV miRNAs and their potential host target genes. Then, an EBV miRNA and host gene regulation network was constructed. Finally, we chose certain of EBV miRNAs and their target genes for directing further validation of EBV miRNA functions in NPC tumorigenesis.Preliminary experimental results:1、EBV miRNA profiles in NPC tumor and nontumor tissues and cell linesThe expression levels of all44mature EBV-encoded miRNAs and2latent protein genes, LMP1and EBNA1, were detected by qPCR in NPC tumor (16samples) and nontumor tissues (5samples) and in5cell lines. Neither EBV miRNAs nor EBV latent protein genes were detected in the nontumor biopsies or in the EBV-negative cell lines, Most of the40EBV miRNAs residing in the BART region and the latent protein genes were highly expressed in14NPC samples and in the EBV-positive cell line. No BHRF1miRNAs were detected in the nasopharyngeal epithelium, tumor tissues, or non-tumor tissues. In contrast, lymphoma cells derived from human and marmoset, Raji and B95-8, expressed high levels of BHRF1miRNAs but low levels of BART miRNAs。2、Prediction of target genes of EBV miRNAsAs all of BHRF1miRNAs were unexpressed in NPC tissues. Two bioinformatic tools, TargetScan and RepTar, were used to predict target genes of all of BART miRNAs. A total of5,569human target genes were predicted by TargetScan, and6,494genes were predicted by RepTar. A total of3,140genes were identified by both bioinformatic tools. Among these3,140genes, BART-18-5p had the highest number of targets (148genes), whereas BART-8, BART-13*, and BART19-3p had no potential target host gene. Then, these3,140target genes were subjected to a functional clustering analysis using DAVID. It appears that BART miRNAs target a wide range of pathways; the top5significant pathways include axon guidance, pathways in cancer, the Wnt signaling pathway, regulation of the actin cytoskeleton, and adherens function have found.3、Integrated analysis of the EBV targetome and the transcriptome of NPCBecause the predicted EBV miRNA target genes may not be expressed in epithelial tissues and miRNAs are negative regulators of mRNAs in most circumstances, an elevated expression of EBV miRNAs is expected to cause downregulation of the miRNA target genes. To determine how EBV miRNAs control the expression of host genes, we analyzed the mRNA expression profile of NPC and determined whether the downregulated genes were potential targets of EBV miRNAs. The SAM test was applied to compare the NPC and nontumor tissue transcriptomes. We found that732genes were significantly dysregulated in NPC;270genes were upregulated, while462genes were down-regulated. The462down-regulated genes were compared to the above-mentioned3,140potential EBV miRNA target genes. We found105 genes that were both downregulated in NPC and predicted as potential targets of EBV miRNAs by both TargetScan and RepTar.4、EBV miRNA and target gene regulation networkFor the105genes that are downregulated in NPC and targeted by EBV miRNAs, we identified175miRNA:mRNA target pairs. We identified28EBV miRNAs that target at least one of these105genes. BART-22has the highest number of targets (37genes), followed by BART-8*(23target genes), BART-18-5p (12), BART-9(11), and BART-14(11), these top5EBV miRNAs regulated94of the175(53.4%) miRNA:mRNA target pairs. Similarly, some genes, such as ATRX, BTRC, CTBP2, FOXP1, and NFIB, were predicted to have several EBV miRNA binding sites in their3’-untranslated regions. To illustrate the complexity of the regulatory relationships between EBV miRNAs and host genes, their regulation network were visualized using Pajek.5、Preliminary validation of EBV-miRNA-BART10targeting BTRCOur previous data showed the WNT signaling pathway was abnormally activated in NPC, and its abnormally activation was closely related to EBV infectio. A key molecule in WNT signaling pathway, BTRC, also belonged to the462down-regulated genes in NPC by SAM analysis in our previous data. In this paper, BTRC was predicted as a target gene of some EBV miRNAs usinge two bioinformatic tools. Through the preliminary experiments, we found that EBV-miRNA- BART-10was the most closely connected with BTRC, so EBV-miRNA-BART-10and BTRC were selected for further validation. Real-time PCR data showed that EBV-miRNA-BART-10was upregulated and negative correlated with BTRC gene expression in NPC clinical samples. The real-time PCR and western blot were performed to confim that BTRC was down-regulated by EBV miRNA-BART-10mimics in mRNA and protein level. The luciferase assay showed that EBV-miRNA-BART-10mimics down-regulated BTRC luciferase reporter expression, suggesting that EBV-miRNA-BART-10could bind to3’-UTR of BTRC and reduce BTRC expression. The downexpression of BTRC may be caused by EBV-miRNA-BART-10regulation in NPC.Here, we established a comprehensive EBV miRNAs profile for the first time, which providing useful clues for directing further understanding of EBV miRNAs function in NPC. Second, construction on the EBV miRNAs and host gene regulation network offered an important reference between their relationships for further study. Finally, BTRC, as a critical molecule in WNT signaling pathway, was confirmed that it was regulated by EBV-miRNA-BART-10mimics, we hypothesized that EBV-miRNA-BART-10was likely to participate in NPC through the regulation of BTRC.