Aberrant methylation-mediated silencing of microRNAs contributes to HPV-induced anchorage independence
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Saskia M. Wilting1, Viktorian Miok1,2,*, Annelieke Jaspers1,*, Debby Boon1, Hanne Sørgård1, Malin Lando3, Barbara C. Snoek1, Wessel N. van Wieringen2, Chris J.L.M. Meijer1, Heidi Lyng3, Peter J.F. Snijders1, Renske D.M. Steenbergen1
1Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands
2Department of Epidemiology & Biostatistics, VU University Medical Center, Amsterdam, The Netherlands
3Department of Radiation Biology, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
*These authors have contributed equally to this work
Renske D.M. Steenbergen, email: email@example.com
Keywords: anoikis, cervical cancer, DNA methylation, CIN lesion
Received: March 30, 2016 Accepted: May 13, 2016 Published: May 30, 2016
Cervical cancer and a subset of anogenital and head-and-neck carcinomas are caused by high-risk types of the human papillomavirus (hrHPV). During hrHPV-induced malignant transformation keratinocytes become able to grow anchorage independently, a tumorigenic trait at least partly associated with inactivation of tumor suppressor genes. We used hrHPV-containing keratinocytes to investigate the role of DNA methylation-mediated silencing of microRNAs (miRNAs) in the acquisition of anchorage independence.
Anchorage dependent (n=11) and independent passages (n=19) of 4 hrHPV-immortalized keratinocyte cell lines were treated with 2′-deoxy-5-azacytidine (DAC). Genome-wide miRNA expression profiles before and after treatment were compared to identify miRNAs silenced by methylation. Bisulfite sequencing and methylation-specific PCR showed increased methylation of hsa-mir-129-2/-137/-935/-3663/-3665 and -4281 in anchorage independent HPV-transformed keratinocytes and cervical cancer cell lines. Mature miRNAs derived from hsa-mir-129-2/-137/-3663 and -3665 showed functional relevance as they decreased anchorage independence in cervical cancer cell lines. Cervical (pre)cancerous lesions demonstrated increased methylation of hsa-mir-129-2/-935/-3663/-3665 and -4281, underlining the clinical relevance of our findings.
In conclusion, methylation-mediated silencing of tumor suppressive miRNAs contributes to acquisition of an anchorage independent phenotype. This study further substantiates the importance of miRNAs during early stages of carcinogenesis and underlines their potential as both disease markers and therapeutic targets.
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