RESEARCH ARTICLE Effects of Methylation Status of CpG Sites within the HPV16 Long Control Region on HPV16-Positive Head and Neck Cancer Cells

Objective To map comprehensively the methylation status of the CpG sites within the HPV16 long control region (LCR) in HPV-positive cancer cells, and to explore further the effects of meth-ylation status of HPV16 LCR on cell bioactivity and E6 and E7 expression. In addition, to analyze the methylation status of the LCR in HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) patients. Methods and Materials Methylation patterns of HPV16 LCR in UM-SCC47, CaSki, and SiHa cells and HPV16-posi-tiive OPSCC specimens were detected by bisulfite-sequencing PCR and TA cloning. For cells treated with 5-aza-20-deoxycytidine and E6 and E7 knockdown, MTS and trypan blue staining, annexin-V and 7-AAD staining, and prodidium iodide were used to evaluate cell growth and cell proliferation, cell apoptosis, and cell cycle arrest, respectively. E6 and E7 mRNA and protein expression were analyzed by quantitative real-time PCR and immunocy-tochemistry, respectively