人類肝癌組織中粒線體DNA變異之研究

粒線體是生物能量的來源，在細胞的能量代謝上扮演了主要的角色。近年來許多粒線體DNA突變已被發現並且與人類的各種疾病有著關聯性。而粒線體功能的缺失也被認為與癌症有關，然而在癌化過程中粒線體DNA的改變目前仍不十分清楚。肝癌(hepatocellular carcinoma)是台灣最常見的癌症之一，為了瞭解粒線體DNA在肝癌癌化過程中所扮演的角色，本論文利用定序及競爭性PCR方法，檢測了35位肝癌患者之腫瘤及相對應之非腫瘤肝組織在粒線體DNA突變、拷貝數的改變及斷損突變等的相關情形。過去的研究顯示：粒線體DNA D-loop 區域是發生體突變的好發點，本論文的結果顯示有45.7 % (16/35) HCC含有D-loop 區域體突變且多為同質性。其中有56.3 % (9/16)的突變是T→C 及G→A核苷酸置換，而有56.3 % (9/16)是發生在CSB II區核苷酸位置(np) 303處的單一點突變。除此之外，與相對應之非腫瘤肝組織比較，有55.9 % (19/34) HCC其粒線體DNA拷貝數明顯下降；同時具有體突變又有拷貝數下降情形之HCC佔62.5 % (10/16)，而沒有體突變但是有拷貝數下降情形之HCC也有50 % (9/18)。相反地，在HCC患者之非腫瘤肝組織中，粒線體DNA 4977 bp 斷損突變之發生率有48.6 % (17/35)，而腫瘤組織只有5.7 % (2/35) 發生斷損突變。相似的結果，在HCC患者之非腫瘤肝組織中，粒線體DNA多重斷損突變的發生率亦是遠高於腫瘤組織 (2/35)。為了進一步確認此一突變確實是粒線體DNA的多重斷損突變，利用選殖技術與定序分析，結果發現在非腫瘤肝組織中有兩個大片段的斷損突變，其一是7,767 bp斷損突變，另一則是7,438 bp斷損突變。總之，在肝癌部份的肝組織其大片段的斷損突變發生率較低，可能顯示在癌化過程中帶有大片段斷損突變的粒線體有一選擇性機制的存在。 綜合以上實驗結果可以發現粒線體基因的不穩定性導致的突變可能參與在肝癌的癌化過程。

Mitochondrion is a source of cell energy and it plays a central role in cellular metabolism. In recent years, many mutations in mitochondrial DNA (mtDNA) have been characterized and were correlated with several human diseases. Defects in mitochondrial function have also been proposed to be associated with carcinogenesis. However, the role of mtDNA alterations in carcinogenesis still remains unclear. Hepatocellular carcinoma (HCC) is one of the most common cancers in Taiwan. To understand the role of mtDNA in hepatocarcinogenesis, the somatic mutation, the change of copy number and the deletions of mtDNA were examined in HCC and corresponding non-tumorous liver tissues of 35HCCs patients by direct sequencing and competitive PCR in this thesis. The D-loop region was found to be a hot spot for somatic mutation in mtDNA of the tumors. The results revealed that 45.7 % (16/35) of the HCCs carried somatic mutation in the D-loop region, and the most mutations are homoplasmy. Moreover, 56.3 % (9/16) of these mutations were T-to-C and G-to-A transitions and also 56.3 % (9/16) of them were located in the CSB II region at nucleotide position (np) 303， which has single point mutation of mtDNA. In addition, compared with corresponding non-tumor tissues, the mtDNA copy number of HCCs was significantly decreased in 55.9 % (19/34) of the patients with HCC. In those with somatic mutation in the D-loop of mtDNA, not only 62.5 % (10/16) of the HCCs have mtDNA depletion but also 50 % (9/18) of the HCCs without somatic mutation in the D-loop region had a reduced copy number of mtDNA. In contrast, the frequency of mtDNA 4977 bp deletion (the common deletion in mtDNA) was 48.6 % (17/35) in corresponding non-tumorous tissues, and only 5.7 % (2/35) of mtDNA 4977 bp deletion in HCCs. Moreover, the incidence of multiple deletions f mtDNA in the corresponding non-tumorous tissue was significantly higher than that in the HCCs (2/35). In order to confirm that multiple deletions occurred in the mtDNA, two large-scaled deletions of mtDNA in the corresponding non-tumorous tissue were cloned and sequenced. One is a 7,767 bp deletion and the other is a 7,438 bp deletion of mtDNA. The lower frequency of the large deletions in cancerous tissue maybe indicate a selective mechanism against mitochondria harbored large deletions. Taken together, these observations suggest the mtDNA instability might be involved in the hepatocarcinogenesis.