5azadC treatment upregulates miR-375 level and represses HPV16 E6 expression
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Adrien Morel1, Aurélie Baguet1, Jérôme Perrard1, Caroline Demeret2, Elise Jacquin3, David Guenat4,5, Christiane Mougin1,4 and Jean-Luc Prétet1,4
1EA3181, Université Bourgogne Franche-Comté, LabEx LipSTIC ANR-11-LABX-0021, Besançon, France
2Département de Virologie, Institut Pasteur, Unité de Génétique Moléculaire des Virus à ARN, CNRS UMR 3569, Université Paris Diderot, Sorbonne Paris Cité, Paris, France
3Signalling Department, The Babraham Institute, Babraham Research Campus, Cambridge, United Kingdom
4Centre Hospitalier Régional Universitaire, Besançon, France
5Department of Medicine, Division of Oncology, Stanford Cancer Institute, Stanford University, Stanford, California, USA
Jean-Luc Prétet, email: [email protected]
Keywords: HPV, DNA methylation, E6, miRNA, 5azadC
Received: August 20, 2016 Accepted: April 10, 2017 Published: May 02, 2017
High-risk human papillomaviruses are the etiological agents of cervical cancer and HPV16 is the most oncogenic genotype. Immortalization and transformation of infected cells requires the overexpression of the two viral oncoproteins E6 and E7 following HPV DNA integration into the host cell genome. Integration often leads to the loss of the E2 open reading frame and the corresponding protein can no longer act as a transcriptional repressor on p97 promoter. Recently, it has been proposed that long control region methylation also contributes to the regulation of E6/E7 expression.
To determine which epigenetic mechanism is involved in HPV16 early gene regulation, 5-aza-2′-deoxycytidine was used to demethylate Ca Ski and SiHa cell DNA. Decreased expression of E6 mRNA and protein levels was observed in both cell lines in an E2-independent manner. E6 repression was accompanied by neither a modification of the main cellular transcription factor expression involved in long control region regulation, nor by a modification of the E6 mRNA splicing pattern. In contrast, a pronounced upregulation of miR-375, known to destabilize HPV16 early viral mRNA, was observed. Finally, the use of miR-375 inhibitor definitively proved the involvement of miR-375 in E6 repression. These results highlight that cellular DNA methylation modulates HPV16 early gene expression and support a role for epigenetic events in high-risk HPV associated-carcinogenesis.
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