Identification of HPV16-p16INK4a mediated methylation in oral potentially malignant disorder.

ABSTRACT

To evaluate the possible involvement of epigenetic modulation by HPV16-p16INK4a in oral potentially malignant disorder (OPMD). We generated DNA-methylation profiles, according to p16INK4a expression and HPV16 genotype (positive or negative), of OPMD samples and p16INK4a-HPV16 negative samples (used as control), using reduced-representation bisulphite sequencing (RRBS-Seq- Illumina) technology. Twelve samples, four for each group, as follows 1) p16INK4a+ HPV16+; 2) p16INK4a+ HPV16-; 3) p16INK4a- HPV16-, were analysed in triplicate for DNA-methylation profiles. Fifty-four per cent of DMRs were hypermethylated and 46% were hypomethylated. An increase in methylation of loci in OPMD was independent of the presence of HPV. The hypermethylated genes in HPV+ samples were associated with signalling pathways such as NICD traffics to nucleus, signalling by NOTCH1 (p = 0.008), Interferon-gamma (p = 0.008) and Interleukin-6 signalling (p = 0.027). The hypomethylated genes in HPV infection were associated with TRAF3-dependent IRF activation pathway (p = 0.002), RIG-I/MDA5 mediated induction of IFN-alpha/beta pathways (p = 0.005), TRAF6 mediated IRF7 activation (p = 0.009), TRIF-mediated TLR3/TLR4 signalling (p = 0.011) and MyD88-independent cascade release of apoptotic factors (p = 0.011). Protein association analysis of DMRs in OPMD revealed 19 genes involved in the cell cycle regulation, immune system, and focal adhesion. Aberrantly methylated loci in OPMD were observed in p16INK4a positive samples which suggests that a shift in global methylation status may be important for cancer progression. The results suggest that HPV infection in OPMD induces modulation of genes related to the immune system and regulation of the cellular cycle.