Aberrant methylation-mediated silencing of microRNAs contributes to HPV-induced anchorage independence

Abstract

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.

miR-144 inhibits growth and metastasis of cervical cancer cells by targeting VEGFA and VEGFC

“…In cervical cancer, DNA methylation-mediated silencing of a rapidly growing number of coding and non-coding genes has been identified and their functional relevance has been demonstrated (32). Increased methylation has been demonstrated for a number of miRNAs, including miR-34b, miR-95, miR-124 and miR-375 (32). The present study found that miR-144 expression was also repressed, indicating that DNA methylation-mediated silencing of miR-144 may exist in cervical cancer.…”

Identification of Deregulated Pathways, Key Regulators, and Novel miRNA-mRNA Interactions in HPV-Mediated Transformation

“…Copy number changes, mRNA, and miRNA expression were determined in all four cell lines at six to eight different passages (time points) ( Table 1). As described previously, the selected time points represent distinct stages in HPV-induced transformation ( Figure 1a) [16]. Table 1.…”

“…Table 1. Passage numbers included for all four HPV-transformed cell lines [16]. FK16A FK16B FK18A FK18B T1 p18 p21 p19 p17 T2 p22 p22 p21 p18 T3 p39 p45 p47 p40 T4 p52 p51 p60 p52 T5 p109 p89 p92 p90 T6 p115 p102 p99 p98 T7 p206 p140 p148 p146 T8 p222 p169 p160 p164 FK16A and FK16B contain HPV16, whereas FK18A and FK18B contain HPV18.…”

“…These are urgently needed to improve current cervical cancer screening programs for the coming era, especially with the implementation of HPV-based screening and HPV vaccination in more and more countries. [16]. MiRNA microarrays of cell line FK18A at T4 and T5 did not pass quality control and were therefore excluded from miRNA analysis.…”

“…MiRNA microarrays of cell line FK18A at T4 and T5 did not pass quality control and were therefore excluded from miRNA analysis. Unsupervised hierarchical cluster results based on (b) DNA copy number, (c) overall mRNA expression, and (d) overall miRNA [16] expression are shown for FK16A, FK16B, FK18A, and FK18B.…”