Abstract
BackgroundAberrant methylation is a frequent event in oral cancer.MethodsIn order to better characterize these alterations, a search for genes downregulated by aberrant methylation in oral squamous cell carcinoma (OSCC) was conducted through the mining of ORESTES dataset. Findings were further validated in OSCC cell lines and patients’ samples and confirmed using TCGA data. Differentially expressed genes were identified in ORESTES libraries and validated in vitro using RT-PCR in HNSCC cell-lines and OSCC tumor samples. Further confirmation of these results was performed using mRNA expression and methylation data from The Cancer Genome Atlas (TCGA) data.ResultsFrom the set of genes selected for validation, CA3 and FHL1 were downregulated in 60% (12/20) and 75% (15/20) of OSCC samples, respectively, and in HNSCC cell lines. The treatment of cell lines JHU-13 and FaDu with the demethylating agent 5'-aza-dC was efficient in restoring CA3 and FHL1 expression. TCGA expression and methylation data on OSCC confirms the downregulation of these genes in OSCC samples and also suggests that expression of CA3 and FHL1 is probably regulated by methylation. The downregulation of CA3 and FHL1 observed in silico was validated in HNSCC cell lines and OSCC samples, showing the feasibility of integrating different datasets to select differentially expressed genes in silico.ConclusionsThese results showed that the downregulation of CA3 and FHL1 data observed in the ORESTES libraries was validated in HNSCC cell lines and OSCC samples and in a large cohort of samples from the TCGA database. Moreover, it suggests that expression of CA3 and FHL1 could probably be regulated by methylation having an important role the oral carcinogenesis.
Highlights
Aberrant methylation is a frequent event in oral cancer
Selection of downregulated genes in Head and neck squamous cell carcinoma (HNSCC) The program Blastn was run for 946,260 Open Reading Expressed Sequence Tags (ORESTES) against the Reference Sequences (RefSeq) database of human genes, resulting in 570,214 ORESTES selected in this analysis
The results showed that Carbonic anhydrase III (CA3) was expressed in four of the cell lines evaluated, whereas no Messenger RNA (mRNA) was detected in the JHU-13 cell line, suggesting that this gene is downregulated in this cell line
Summary
Aberrant methylation is a frequent event in oral cancer. Squamous cell carcinoma (SCC) is the most frequent histological subtype of oral cavity cancers This disease originates from the epithelial tissue that covers the entire aero digestive tract and accounts for more than 90% of all malignancies in that anatomical region [1]. This cancer site is among the most common worldwide and a major cause of morbidity and mortality [2]. Several studies in oral carcinogenesis point to an important relationship between aberrant DNA methylation at the promoter of tumor suppressor genes and their inactivation [4,5,6,7,8,9].
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