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 methylation 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
Methylation patterns of HPV16 LCR in UM-SCC47, CaSki, and SiHa cells and HPV16-positiive OPSCC specimens were detected by bisulfite-sequencing PCR and TA cloning. For cells treated with 5-aza-2'-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 immunocytochemistry, respectively.


RESULTS
Hypermethylation status of the LCR in UM-SCC47 (79.8%) and CaSki cells (90.0%) and unmethylation status of the LCR in SiHa cells (0%) were observed. Upon demethylation, the cells with different methylation levels responded differently during growth, apoptosis, and cell cycle arrest, as well as in terms of their E6 and E7 expression. In HPV16-positive OPSCC patients, the methylation rates were 9.5% in the entire LCR region, 13.9% in the 5'-LCR, 6.0% in the E6 enhancer, and 9.5% in the p97 promoter, and hypermethylation of p97 promoter was found in a subset of cases (20.0%, 2/10).


CONCLUSIONS
Our study revealed two different methylation levels of the LCR in HPV16-positive cancer cells and OPSCC patients, which may represent different carcinogenesis mechanisms of HPV-positive cancers cells. Demethylating the meCpGs in HPV16 LCR might be a potential target for a subgroup of HPV16-positive patients with head and neck squamous cell carcinoma.
Department of Otorhinolaryngology, Head and Neck Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, China; Department of Otorhinolaryngology, Head and Neck Surgery, University of the Ryukyus, Okinawa, Japan; Department of Otorhinolaryngology, Head and Neck Surgery, Affiliated hospital of Zunyi Medical University, Zunyi, China.
Chunlin
C
Zhang
Department of Otorhinolaryngology, Head and Neck Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, China; Department of Otorhinolaryngology, Head and Neck Surgery, University of the Ryukyus, Okinawa, Japan.
Zeyi
Z
Deng
Department of Otorhinolaryngology, Head and Neck Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, China; Department of Otorhinolaryngology, Head and Neck Surgery, University of the Ryukyus, Okinawa, Japan; Department of Otorhinolaryngology, Head and Neck Surgery, The First People's Hospital of Guangzhou, Guangzhou, China.
Xiaoli
X
Pan
Department of Otorhinolaryngology, Head and Neck Surgery, University of the Ryukyus, Okinawa, Japan.
Takayuki
T
Uehara
Department of Otorhinolaryngology, Head and Neck Surgery, University of the Ryukyus, Okinawa, Japan.
Mikio
M
Suzuki
Department of Otorhinolaryngology, Head and Neck Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, China.
Minqiang
M
Xie
D017209:Apoptosis / Q000502:physiology; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics*; D006258:Head and Neck Neoplasms / Q000473:pathology / Q000821:virology*; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D009856:Oncogene Proteins, Viral
D009856:Oncogene Proteins, Viral