A minimal DNA 5-methylcytosine signature of oral tongue squamous cell carcinoma [HumanHT-12 V4.0]
ABSTRACT: Oral tongue squamous cell carcinomas (OTSCC) are a homogenous group of aggressive tumors in the head and neck region, with a rising incidence among younger population. The role of altered DNA methylation in OTSCC and its link with clinical parameters has not been fully assessed yet. We performed genome-wide methylation analysis of oral tongue primary tumors (n = 52) using 485, 512 probes and correlated altered methylation with differences in gene expression. We used an ensemble machine-learning algorithm to identify differentially methylated probes and regions predictive of survival, risk habits, nodal status, tumor stage, and HPV infection followed by validation using data from the cancer genome atlas (TCGA) project on oral tongue (n = 24) and tumors from all subsites of head and neck region (n = 50). Whole-genome gene expression profiling was carried out with Illumina HumanHT12 v4 expression BeadChip (Illumina, San Diego, CA) with 21 tumors and their matched adjacent normal tissues. Total RNA was extracted using PureLink RNA mini kit (Invitrogen) and RNA quality was checked on the bioanalyzer using RNA Nano6000 chip (Agilent). RNA samples with poor RIN numbers (≤7) on the bioanalyzer chip, indicating partial degradation of RNA were processed using Illumina WGDASL assay and the ones with good RIN numbers (>7) were labelled using Illumina TotalPrep RNA Amplification kit (Ambion) as per the manufacturer’s recommendations. Targets were used to hybridize arrays and arrays were processed according to the manufacturer’s recommendations. Arrays were scanned using HiScan, Illumina, and the data collected were analyzed with GenomeStudio V2011.1 Gene Expression module 1.9.0 (Illumina) to check for the assay quality control probes. Raw signal intensities were exported from GenomeStudio for transformation, normalization and differential expression analyses using R.