Oxidative Stress-Induced Mitochondrial DNA Methylation Affect Cell Viability

在近代生物遺傳學中發現了一種在不改變DNA序列的狀況下，造成表現型(phenotype) 的改變，並且可以繼續遺傳到子代的新興學問，稱為上位遺傳學（Epigenetics），其包含了DNA甲基化（DNA methylation）、組蛋白修飾（histone modification）、非編碼RNA（non-coding RNA）等。其中DNA甲基化被認為與基因印痕（gene imprinting）、胚胎發展（Embryonic development）、X染色體失活（X-chromosome inactivation）、癌化（oncogenesis）1與基因沉默（gene silencing）有關。而有關於細胞genomic DNA甲基化的研究更是十分的熱門，但卻對於細胞中另一個含有DNA及主要能量生產來源之胞器:「粒線體」的甲基化卻是少有研究。曾有前人研究指出，粒線體DNA的甲基化程度很低，且粒線體DNA甲基化與細胞存活度的關聯也被認為相當微弱。但在2009與2011年分別有文獻證明了粒線體DNA各個區域間有不同的可被甲基化程度與專屬粒線體的DNA甲基轉移酶。且近期由於偵測技術進步，我們認為粒線體的甲基化程度或許也應被重新評估。本研究將bisulfite conversion後的粒線體DNA利用焦磷酸定序法（Pyrosequecing）進行直接且更精確的甲基化偵測，並比較 HeLa 與 PC9 兩種細胞在H2O2所造成的氧化壓力下，粒線體的ATP6、ND1、ND6、TERM以及D-loop 等5個區域甲基化程度的變化。實驗結果顯示HeLa 與PC9在H2O2處理後，PC9顯現出較高的存活率，表示其對於氧化壓力具有較好的抗性與耐受性。在HeLa cells處理H2O2後，各基因甲基化程度並無顯著變化，但TERM與D –loop趨勢呈現上升；PC9 cells在H2O2處理後，5個區域的甲基化程度均是上升的。另一方面，在未處理H2O2的狀態下，HeLa cells甲基化程度皆高於PC9 cells，但經過H2O2處理後則為PC9 cells 較高且差異大。由本次研究結果我們推測，當細胞受到氧化壓力的影響時，會引起粒線體DNA的甲基化，並且不同細胞會有不同程度的差異。另外，我們也推測甲基化的程度會影響細胞在抵抗氧化壓力時的細胞存活率。Epigenetics which is defined as ” A phenomenon that changes the phenotype without changing the underlying DNA sequence, and it can inherit to offspring.” is one of the major research topics in modern biology. In addition, epigenetics includes DNA methylation, histone modification, and non-coding RNA. In which DNA methylation is associated with gene imprinting, embryonic development, X-chromosome inactivation, oncogenesis, and gene silencing. By focusing on DNA methylation, there are lots of researches in nuclear DNA, however, the information about mitochondrial DNA methylation is relatively rare. Nevertheless, it is known that mitochondria play an important role in ATP producing. Previous study shows a low methylation pattern in mitochondria and consider that the relationship between mtDNA methylation and cell viability is weak . But, there were two papers which were published in 2009 and 2011 respectively were focusing on this issue as well. One indicates the different methylated accessibility of mtDNA and the other shows the appearance of mitochondrial DNA methyltransferase 1. Recently, the mitochondrial DNA methylation might need re-evaluation due to the detection technology progress. In this project, one of the major technology, pyrosequencing which is more direct and accurate method to detect oxisative stress-induced mtDNA methylation on HeLa cell and PC9 cell, is applied. By carrying out this technique, five mitochondrial-encode gene: ATP6, ND1, ND6, TERM and D-loop are analysed. Based on our research, PC9 cell has better viability than HeLa cell after H2O2 treatment. Moreover, methylation level of five genes are all increasing in PC9 cell, but no significant difference in HeLa cell. Therefore, baseline data shows that HeLa cell has higher methylation level than PC9 in mtDNA, however, the methylation level of PC9 cell is above than HeLa cell after H2O2 treatment. It is believed that oxidative stress-induced DNA methylation has cell-specificity and the methylation level might affect cell viability