Epstein-Barr Virus Infection Downregulates MicroRNA-203Expression And The Interaction Between Them Is Involved In The Pathogenesis Of Nasopharyngeal Carcinoma

The Epstein-Barr virus (EBV) is one of the most highly transforming viruses and is associated with several malignancies, including lymphatic Burkitt's lymphoma (BL) and nasopharyngeal carcinoma (NPC). Nevertheless, the role of EBV in the development of malignancies, especially in epithelial tumors such as NPC, remains poorly understood. MicroRNAs belong to a class of short, highly conserved noncoding RNAs that are known to suppress the expression of protein-coding genes through imperfect base pairing with the3' untranslated regions (UTRs) of target messenger RNAs (mRNAs). MiRNAs have been implicated in the control of various biological processes, such as cell proliferation, apoptosis, and differentiation. Growing evidence has indicated that deregulation of miRNAs also contributes to tumorigenesis. However, It remains largely to be clarified about the mechanism on how EBV regulates the expression of miRNAs in NPC.Based on previous study, we used miRNA microarray to investigate the differentially expressed miRNAs in293-EBV cell. This approach provided new insight into the interaction between the virus and epithelial cells during cancer development. Furthermore, miR-203is a potential biomarker for the diagnosis and therapy of EBV-associated NPC.[miR-203was down-regulated in EBV infected epithelial cells, lymphocytes and poorly differentiated nasopharyngeal carcinoma tissues]The miRNA profile of293-EBV cells was examined using microarrays. The results showed that there were3miRNAs up-regulated (fold change>2.5) and15miRNAs down-regulated (fold change>2.5) in 293-EBV cell contrasting with EBV negative293cells. Among the downregulated miRNAs, miRNA-203(miR-203) was downregulated approximately3.7-fold. Because miR-203is expressed specifically in epithelial cells and acts as a tumor suppressor in several epithelial cancers, it was chosen for further study. qRT-PCR results revealed that expression levels of miR-203was down regulated in EBV-infected epithelial cell and EBV transformed lymphocytes. Moreover, loss of EBV from the293-EBV cells resulted in an almost complete restoration of miR-203expression. qRT-PCR results revealed that expression levels of miR-203was lower more than4.25-fold in nasopharyngeal carcinoma tissues than in normal nasopharyngeal epithelial tissues purified by frozen section and laser capture microdissection.[miR-203inhibits EBV-induced proliferation and transformation in epithelial cells through regulating the expression of the novel targets, E2F3and CCNG1]The basic information of miR-203was collected from miRBase database, miR-203targets that were predicted by TargetScan and Pictar for further study. The luciferase reporter assay demonstrated that E2F3and CCNG1genes were targets of miR-203. MiR-203can regulate the expression of E2F3and CCNG1mainly at protein level as well as mRNA level. At posttransfection with the miR-203mimics into293-EBV cells, miR-203can inhibit the cell proliferation significantly and the number of cells in S phase decreased, and the number in the G0/G1phase of the cell cycle increased, which demonstrated inhibition of S-phase entry. This change was overcome by overexpression of the target genes, E2F3and CCNG1, with miR-203. Intratumoral injection of miR-203led to a significant decrease in the size of the tumor in nude mice with transplanted tumors, suggesting that miR-203inhibits the tumorigenicity of epithelial cells promoted by EBV infection. Expression of E2F3and cyclin G1decreased after the introduction of miR-203.[LMPl down regulated the expression of miR-203through JNK and NF-κB signal pathway]LMP1and LMP2are EBV protein to be identified as an oncoproteins. miR-203expression was down regulated at both the primary and mature transcript levels in293-EBV and NP69-LMP1cells. However, miR-203expression was not changed obviously in LMP2-overexpressed293and NP69cells. Repression of LMP1significantly increased miR-203expression in both cell lines. The correlation between the expression of LMP1and miR-203was further determined in additional NPC or noncancer specimens. miR-203expression is obvious in the tissues adjacent to NPC tissues where LMPl is not expressed and miR-203expression was downregulated in most areas of the NPC tissues that were positive for LMP1expression. However, some areas in the NPC tissue those were LMP1negative expressed miR-203obviously.There are three C-terminal activating region (CTAR) domains in LMP1:CTAR1,CTAR2and CTAR3. To determine whether the pathways mediated by LMP1are involved in LMP1-driven miR-203downregulation, inhibitors of the NF-κB, JNK, and p38pathways were used. The JNK inhibitor significantly abolished downregulation of miR-203. Additionally, inhibition of NF-κB restored miR-203expression. However, the p38inhibitor had no obvious effect on miR-203expression. The transfection of an expression plasmid containing an LMP1mutant lacking CTAR3had no effect on miR-203expression in the293-EBV and NP69-LMP1cell lines.[miR-203expression is associated with the metastasis of NPC and the antibody level of EBV VCA-IgA in NPC patients] Previous study showed miR-203is downregulated in NPC.miR-203acted as a suppressive gene function in NPC. miRNAs array was used to detect the miRNAs profile and i n situ hybridization (ISH) analysis was used to detect miR-203expression in NPC tissues. Significant and positive correlation was found between miR-203expression and tumor metastasis in NPC. Patients with lower miR-203expression resulted in poor outcome and high rates of metastasis.