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pp.23-32
Immortalization is an essential process of the transformation of cells to a neoplastic growth. High risk human papillomavirus (hrHPV) infection has been the major cause of head and neck squamous cell carcinoma (HNSCC). The aim of this study was to search for a novel pathway causing immortalization in HPV16 E6/E7 transfected immortalized oral keratinocytes (IHOK). hrHPV integration sites were identified through DNA sequencing. HPV16 E6/E7 genes were integrated into 1q32.2, 12q21.2, 15q15.2, and 19q13.43 in IHOKs. Array-CGH was conducted to examine the deranged sites of the genes of IHOK. Of the 587 amplification genes, 70 genes were resided on chromosome 20. We selected PLAGL2 and MAPRE1 as the most amplified genes. PLAGL2 and MAPRE1 mRNA showed higher expression in IHOK than in normal keratinocytes. Knockdown of MAPRE1 significantly reduced telomerase activity. The analysis using a public database substantiated our data, showing the amplification of chromosome 20 and MAPRE1. In conclusion, our results suggest that MAPRE1 could play a crucial role in activating telomerase activity in hrHPV-infected cells. This finding may provide basic data to develop a novel target therapy for hrHPV-related HNSCC.
Ⅰ. INTRODUCTION
II. MATERIAL AND METHODS
1. Cell culture
2. Polymerase Chain Reaction (PCR), Reverse Transcription-PCR (RT-PCR), and real-time PCR
3. Analysis of HPV16 E6/E7 integration sites
4. Array-CGH analysis
5. siRNA transfection
6. Telomeric repeat amplification protocol (TRAP) assay
7. Public database analysis
8. Statistical analysis
III. RESULTS
1. Integration of HPV16 E6/E7 in IHOKs
2. Increased genomic instability in IHOKs
3. MAPRE1 mRNA related to hTERT expression and activity in IHOKs
4. Chromosome 20 instability in public data base
IV. DISCUSSION
CONFLICTS OF INTEREST
REFERENCES
