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

ABSTRACT: Cervical cancer and a subset of anogenital and head-and-neck carcinomas are caused by persistent infection with high-risk types of the human papillomavirus (hrHPV). Early stages of hrHPV-induced carcinogenesis can be faithfully mimicked in vitro. A major hallmark of hrHPV-transformed cells is their ability to grow anchorage independently, an oncogenic trait known to depend on inactivation of tumour suppressor genes. This study used an in vitro model of hrHPV-induced transformation to delineate in a longitudinal manner to what extent DNA methylation-mediated silencing of tumour suppressive microRNAs (miRNAs) contributed to hrHPV-induced anchorage independence. Genome-wide miRNA expression profiles were yielded from anchorage dependent (n=11) and independent passages (n=19) of 4 hrHPV-immortalised keratinocyte cell lines with and without demethylating treatment (DAC). Unsupervised clustering analysis showed that overall miRNA expression patterns discriminated between anchorage dependent and independent cells. Ten miRNA genes potentially silenced by methylation were selected and validated by bisulfite sequencing and methylation-specific PCR. Hsa-mir-129-2, -137, -935, -3663, -3665, and -4281 showed increased methylation in both HPV-transformed keratinocytes and cervical cancer cell lines compared to primary keratinocytes. Mature miRNAs derived from hsa-mir-129-2, -137, -3663, and -3665 decreased anchorage independence in cervical cancer cell lines. Finally, significantly increased methylation of hsa-mir-129-2, -935, -3663, -3665, and -4281 was observed in cervical (pre)cancerous lesions, underlining the clinical relevance of our findings. In conclusion, methylation-mediated silencing of tumour suppressive miRNAs contributes to the acquisition of anchorage independence, supporting the importance of miRNAs during early stages of carcinogenesis and underlining their potential as both disease markers and therapeutic targets.

ORGANISM(S): Homo sapiens

PROVIDER: GSE78277 | GEO | 2017/02/09

SECONDARY ACCESSION(S): PRJNA313085

REPOSITORIES: GEO

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Project description:Little is known about the alterations in microRNA (miRNA) expression patterns during the consecutive stages of cervical cancer development and their association with chromosomal instability and epigenetic changes. We integrated miRNA expression profiles in normal cervical squamous epithelium, high-grade precancerous lesions (CIN2-3), squamous cell carcinomas (SCC) and adenocarcinomas (AdCAs) with previously generated chromosomal profiles of the same samples. In addition, the DNA methylation status of downregulated miRNAs located in a CpG island was determined. Significantly differential expression during the consecutive stages of cervical SCC development was observed for 106 miRNAs. Altered expression of 5 significantly differentially expressed miRNAs, hsa-miR-9 (1q23.2), hsa-miR-15b (3q25.32), hsa-miR-28-5p (3q27.3), hsa-miR-100 and hsa-miR-125b (both 11q24.1) was directly linked to frequent chromosomal alterations. Another 9 significantly downregulated miRNAs were located within a CpG island. Three of these 9 miRNAs, hsa-miR-203, hsa-miR-572, and hsa-miR-638, showed increased methylation in cervical cancer cell lines and HPV-immortalised cells compared to primary keratinocytes. Functional analyses were performed for hsa-miR-9, representing a potential oncogene with increased expression linked to a chromosomal gain, and hsa-miR-203, representing a potential tumour suppressor gene silenced by DNA methylation. Hsa-miR-9 and hsa-miR-203 were found to alter cell viability and anchorage independent growth in vitro, respectively, supporting their oncogenic and tumour suppressive function in cervical cancer. In conclusion, differential expression of 106 miRNAs, partly associated with chromosomal alterations and epigenetic changes, was observed during cervical SCC development. Altered expression of hsa-miR-9 and hsa-miR-203 was shown to be functionally relevant, underlining the importance of deregulated miRNA expression in cervical carcinogenesis. 10 squamous cell carcinomas of the cervix, 9 adenocarcinomas of the cervix, 18 high-grade cervical intraepithelial neoplasias (CIN2-3), and 10 cervical squamous epithelial samples with normal histology were analysed using single channel (Cy3) miRNA microarrays from Agilent (G4471A-016436).

Project description:Cervical cancer results from the accumulation of (epi)genetic aberrations following persistent infection with high-risk human papillomavirus (HPV). In order to define genetic aberrations associated with cervical carcinogenesis, chromosomal profiles of high-grade cervical intraepithelial neoplasia (CIN) were generated. Common aberrations usually encompass large genomic regions and contain numerous genes, hampering identification of actual driver genes. Consequently, direct evidence of chromosomal alterations actively contributing to cervical carcinogenesis has been lacking so far. By analyzing 60 high-grade CIN with high resolution arrayCGH we identified focal chromosomal aberrations that each harbour only one or a few genes. In total 74 focal aberrations were identified encoding 305 genes. Analysis of genes located within these focal aberrations, using two independent expression microarray datasets, revealed concurrent altered expression in high-grade CIN and/or cervical carcinomas compared to normal cervical samples for 8 genes: ATP13A3, HES1, OPA1, HRASLS, EYA2, ZMYND8, APOBEC2 and NCR2. Gene silencing of EYA2, located within a focal gain at 20q13, significantly reduced viability and migratory capacity of HPV16-transformed keratinocytes. Interestingly, for hsa-miR-375, located within the most frequently identified focal loss at 2q35, a direct correlation between a (focal) loss and significantly reduced expression was found. Down-regulation of hsa-miR-375 expression during cervical carcinogenesis was confirmed in a second independent series of cervical tissues. Moreover, ectopic expression of hsa-miR-375 in 2 cervical carcinoma cell lines reduced cellular viability. In conclusion, our data provide a proof of concept that chromosomal aberrations are actively contributing to HPV-induced carcinogenesis and identify EYA2 and hsa-mir-375 as oncogene and tumor suppressor gene, respectively. DNA from microdissected tissues: 60 samples total. 11 high-grade CIN, <5yr preceding hrHPV infection, 43 high-grade CIN >5yr preceding hrHPV infection, 6 CIN3 adjacent to SCC

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

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