DNA Methylation Changes in Clinical Stages of Oral Cancer Progression and Correlation with DOC2B Promoter Hypermethylation

The well-established correlation between global DNA hypomethylation and genomic instability offers one of the mechanistic explanations on how hypomethylation may contribute to carcinogenesis. In our study, levels of 5-methyl -2’-deoxycytidine in oral cancer tissues and microdissected DNA analysed by reverse phase – high performance liquid chromatograp hy (RP-HPLC) revealed a generalized decrease in all oral cancer samples (1.63 ± 0.16) compared to its matched normal counte
rpart (3.67 ± 0.17; P 0.001). Validation of global DNA methylation as ana lysed by immunohistochemistry (IHC) in successive stages of oral cancer progression in tissue sections using antibody to 5- methyl cytosine revealed reduced immunostaining in dysplasia and oral squamous cell carcinomas (OSCC) compared to normal oral mucosal epithelium. Methylation sensitive arbitrarily primed polymerase chain reaction (MS-AP-PCR) identified se veral non-canonical CpG rich fragments including promoter region of DOC2B ( Double C2 like Domain B) gene spanning -376bp to + 36bp. This was found to be methylated in oral cancer tissues, confirmed by methylation sensitive dimethyl sulfoxide polymerase chain reaction (MS-DMSO-PCR) and validated by bisu lfite genome sequencing (BGS). DOC2B promoter and other differentially methylated sequences identified in our stud y may serve as potential diagnostic markers to delineate human oral cancer and suggests oral cancer patho genesis to altered epigenetic mechanisms.