Epstein-Barr virus-encoded microRNA targets PUMA to promote tumor cell survival

Abstract

INTRODUCTION. Epstein-Barr virus (EBV) is a γ-herpesvirus that is constantly associated with nasopharyngeal carcinoma (NPC). EBV is the first human virus found to express microRNAs (miRNAs), but the functions of these viral miRNAs are largely unknown. Here, we identified and characterized a cellular target of an EBV miRNA known as miR-BART5, which is conserved with rhesus lymphocryptovirus miR-rL1-8 and abundantly expressed in NPC cells. METHOD. miRanda and RNAhybrid were used to predict the potential targets of miR-BART5. Luciferase reporter assay was performed to verify the target site. Gain and loss of miR-BART5 function were interrogated for influence on the expression of target gene. Target expression in EBVassociated NPC tissue samples was also examined. Apoptosis assays were conducted to determine the biological function of miR-BART5 and its target gene in EBV-positive tumor cells. RESULTS. By in silico analysis and screening with luciferase reporter assays, we showed that miR-BART5 targets the 3′-untranslated region of a cellular mRNA transcript encoding p53 upregulated modulator of apoptosis (PUMA). The target site of miR-BART5 was identified and characterized. Regulation of the endogenous PUMA expression was verified by overexpressing miRBART5. In addition, an anti-miR-BART5 oligonucleotide inhibitor was used to confirm that reduction of endogenous miR-BART5 activity in NPC cells led to alteration in PUMA expression. Consistent with a role in the development of NPC, PUMA was found to be significantly underexpressed in about 60% of human NPC tissues constitutively harboring EBV. More importantly, miRBART5-expressing NPC cells were less sensitive to proapoptotic agents adriamycin and etoposide. In addition, apoptosis can be induced in these cells by inhibiting miR-BART5 activity. DISCUSSION. In this work we sought to investigate the function of an EBV miRNA in pathogenesis, particularly in establishing latency in epithelial cells. PUMA was characterized to be a target of miR-BART5. PUMA is a proapoptotic protein belonging to the “Bcl-2 homology 3 domain (BH3)-only” group of the Bcl-2 family. Although it is an immediate downstream target of p53, PUMA can also induce p53-independent apoptosis in response to a large variety of stimuli, including serum starvation, cytokine withdrawal, kinase inhibitors, phorbol esters, glucocorticoids, ischemia-reperfusion, endoplasmic reticulum stress, proteosome inhibition, HIV envelope glycoprotein complex, p73 and FOXO3a. It is not surprising that PUMA is implicated in our work as a critical factor in the prevention of apoptosis in EBV-positive NPC cells. Our findings suggest a model for viral promotion of host cell survival in which EBV encodes an miRNA to repress the expression of PUMA and consequently facilitate the establishment of stable latent infection. ACKNOWLEDGMENT. This work was supported in part by Fogarty International Center of National Institutes of Health (R01 TW06186-01). REFERENCES 1. Pfeffer, S., Zavolan, M., Grasser, F.A., Chien, M., Russo, J.J., Ju, J., John, B., Enright, A.J., Marks, D., Sander, C., and Tuschl, T. (2004) Science 304, 734-736 2. Cai, X., Schafer, A., Lu, S., Bilello, J.P., Desrosiers, R.C., Edwards, R., Raab-Traub, N., and Cullen, B.R. (2006) PLoS Pathog. 2, e23 3. Nakano, K., and Vousden, K.H. (2001) Mol. Cell 7, 683-694 4. Yu, J., Zhang, L., Hwang, P.M., Kinzler, K.W., and Vogelstein, B. (2001) Mol. Cell 7, 673-68