5azadC treatment upregulates miR-375 level and represses HPV16 E6 expression

Abstract

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.

DNA demethylation agent 5azadC downregulates HPV16 E6 expression in cervical cancer cell lines independently of TBX2 expression

“…In this study, we confirmed that the treatment of HPV16 positive cancer cell lines with 5azadC leads to the downregulation of E6 at both mRNA and protein levels (7)(8)(9). As expected, a p53 and p21 restoration was observed in treated cells confirming the functional loss of E6.…”

“…The downregulation of E6 was partly due to the upregulation of miR-375 (8,9), known to target the early HPV16 transcripts (10). Indeed, E6 expression was only partially restored by miR-375 inhibitor in 5azadC-treated cells (9).…”

“…Interestingly, E6 and E7 expression are also regulated by epigenetic mechanisms, and methylation level on viral promoter is higher in HPV-associated cancers than in precancerous lesions or in normal tissues (6). Recently, studies revealed that E6 and E7 expression was downregulated following treatment of HPV positive cancer cell lines by 5aza-2'-deoxycytidine (5azadC), a DNA methyl transferase (DNMT) inhibitor also named decitabine (7)(8)(9). The downregulation of E6 was partly due to the upregulation of miR-375 (8,9), known to target the early HPV16 transcripts (10).…”

“…Recently, studies revealed that E6 and E7 expression was downregulated following treatment of HPV positive cancer cell lines by 5aza-2'-deoxycytidine (5azadC), a DNA methyl transferase (DNMT) inhibitor also named decitabine (7)(8)(9). The downregulation of E6 was partly due to the upregulation of miR-375 (8,9), known to target the early HPV16 transcripts (10). Indeed, E6 expression was only partially restored by miR-375 inhibitor in 5azadC-treated cells (9).…”

Human Papillomavirus E6/E7 and Long Noncoding RNA TMPOP2 Mutually Upregulated Gene Expression in Cervical Cancer Cells

“…miR-375 and miR-139 targeted HPV E6/E7 mRNA in cervical cancer cells. As reported, the mRNA of the HPV E6/E7 gene was targeted by multiple microRNAs, including miR-375 (25,26) and miR-139 (27), in cervical cancer cells. To confirm the targeting effect, mimics of miR-375 were transfected into HeLa cells.…”