Methylation-mediated transcriptional repression of microRNAs during cervical carcinogenesis

Abstract

Deregulated expression of microRNAs (miRNAs) is common and biologically relevant in cervical carcinogenesis and appears only partly related to chromosomal changes. We recently identified 32 miRNAs showing decreased expression in high-grade cervical intraepithelial neoplasia (CIN) and carcinomas not associated with a chromosomal loss, 6 of which were located within a CpG island. This study aimed to investigate to what extent these miRNAs are subject to DNA methylation-mediated transcriptional repression in cervical carcinogenesis.

 

Methylation-specific PCR (MSP) analysis on a cell line panel representing different stages of human papillomavirus (HPV) induced transformation revealed an increase in methylation of hsa-miR-149, -203 and -375 with progression to malignancy, whereas expression of these miRNAs was restored upon treatment with a demethylating agent. All three miRNAs showed significantly increased levels of methylation in cervical carcinomas, whereas methylation levels of hsa-miR-203 and -375 were also significantly increased in high-grade CIN. A pilot analysis showed that increased hsa-miR-203 methylation was also detectable in HPV-positive cervical scrapes of women with high-grade CIN compared with controls. Similar to recent findings on hsa-miR-375, ectopic expression of hsa-miR-203 in cervical cancer cells decreased both the proliferation rate and anchorage independent growth. We found evidence for methylation-mediated transcriptional repression of hsa-miR-149, -203 and -375 in cervical cancer. Methylation of the latter two was already apparent in precancerous lesions and represent functionally relevant events in HPV-mediated transformation. Increased hsa-miR-203 methylation was detectable in scrapes of women with high-grade CIN, indicating that methylated miRNAs may provide putative markers to assess the presence of (pre)cancerous lesions.

Keywords: :

• microRNA
• squamous cell carcinoma
• DNA methylation
• CIN lesion
• HPV
• MSP

Disclosure of Potential Conflicts of Interest

Dr RDM Steenbergen, Prof Dr PJF Snijders and Prof Dr CJLM Meijer are stockholders of Self-screen BV, The Netherlands. All other authors have no conflicts of interest to declare.

Acknowledgments

This research was funded by grants from the VU University Medical Center-Cancer Center Amsterdam (VUMC-CCA) and the Dutch Cancer Society (KWF2010-4668). The authors are grateful to Marlon Lindenbergh-Van der Plas, Tim Schutte, Annina van Splunter, Renée Overmeer, Suzanne Snellenberg, and Sylvia Duin for excellent technical assistance.