| Summary: | 碩士 === 國立成功大學 === 口腔醫學研究所 === 92 === Oral cancer is the fifth leading cause of cancer death in Taiwanese male population. In a previous study, a quantitative alteration of deleted mtDNA 4977bps deletion was detected in the progression of oral carcinogenesis. The displacement loop (D-loop) of mitochondria genome plays important role in mtDNA replication and transcription. A high mutation rate within the region has been reported in other tumors. In the present study, we analyzed mutations in the D-loop region of mtDNA using laser microdissected paired normal, cancer and precancer cells from patients and sixteen cell lines established from cancers of different clinical stages and precancer as well as primary cultured normal oral keratinocytes (hNOK). Quantitative real-time PCR (QRT-PCR) was performed to analyze mtDNA copy number reference to the nuclear DNA. The results indicate a significant lower mutation frequency of the D-loop region in the cell lines compared to the specimens. The hot regions for mutations was distributed between the nucleotide position (np) 54~354 (heavy strand) and 154~454 (light strand) in the cell lines, while microdissected cancer tissues harbored mutations between np134~504 (heavy strand). Ten of fifteen oral cancer cell lines had decreased mtDNA copy number compared with primary cultured hNOK, while the precancer line (DOK) exhibited slightly increased mtDNA copy number between normal and the cancer cell lines. In the clinical samples, significant decreased mtDNA copy number was also observed in cancers compared to normal and precancer tissues. A significant inverse association was found between mtDNA copy number and the D-loop mutation rate in the clinical samples (p<0.01). In conclusion, D-loop mutations were accumulated among mtTFA binding sites and the heavy strand replication origin during oral carcinogenesis. Precancer lesions had a higher mutation rate and broader range of mutation distribution than the cancer tissues did. Oral cancer cell lines presented significantly lower mutation rate compared with those of the clinical specimens. The D-loop mutation rate is inversely associated with mtDNA copy number in cancer tissues. In oral cancer cell lines and clinical specimens, there was a reverse relationship between the 4977bp deletion and copy number of mtDNA. The level of 8-OHdG DNA damage was inversely associated with mtDNA copy number in the cancer cell lines. The mutation frequency of mtDNA D-loop region was also associated with the level of 8-OHdG content in mtDNA. In summary, this study revealed an association between mtDNA D-loop region mutations and oral carcinogenesis progression. The mutation frequency was associated with the mtDNA 8-OHdG oxidative damage and may decrease the mtDNA replication efficiency. The large scale mtDNA deletion was also associated with the decrease in the mtDNA copy number. However, the mechanism for D-loop mutations in oral carcinogenesis progression and their functional effects on the mtDNA replication and cancer development remained to be elucidated.
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