Development of an in vitro system to study the effects of methylenetetrahydrofolate reductase suppression on folate metabolism.

Abstract

遺傳缺陷或維生素葉酸缺乏可能會造成單碳代謝的擾亂而促使疾病發生。人類亞甲基四氫葉酸還原酶基因 (methylenetetrahydrofolate reductase, MTHFR) 之多型性普遍存在。研究指出在葉酸充分的狀態下，此酶 (MTHFR) 之遺傳變異型可以緩解某些癌症的形成。但葉酸缺乏時，此變異型反而是某些癌症危險因子。目前機制尚不清楚。 本論文中藉由慢病毒介導的RNA干擾(RNAi)技術來干擾亞甲基四氫葉酸還原酶 (MTHFR) 的表現，以研究亞甲基四氫葉酸還原酶 (MTHFR) 和環境中葉酸營養因素的交互作用，並探討亞甲基四氫葉酸還原酶 (MTHFR) 與肝癌細胞之細胞週期分佈、葉酸代謝及低亞甲基四氫葉酸還原酶 (MTHFR) 酵素活性是否影響肝癌細胞對於環境中葉酸營養狀況的耐受性。 在人類肝癌細胞干擾亞甲基四氫葉酸還原酶 (MTHFR) 表現之系統模型中，我們發現當亞甲基四氫葉酸還原酶 (MTHFR)之表現受到干擾後其基因、蛋白及酵素活性皆受到抑制，且可能影響肝癌細胞HepG2細胞週期、葉酸代謝能力及影響肝癌細胞對於環境中葉酸營養狀況的耐受性。 此結果證明此酶之表現和葉酸營養狀況密切相關。同時我們認為此種調控機制可能是亞甲基四氫葉酸還原酶 (MTHFR)基因變異影響癌症發生過程的原因之一。 本論文提出亞甲基四氫葉酸還原酶 (MTHFR)與肝癌細胞的生長狀況及對於環境中葉酸營養狀況的反應密切相關，藉由此系統的建立提供未來癌症治療之參考與基礎。

Genetic predisposition or folate deficiency may cause disruption in one-carbon metabolism, which induces disease. The human methyltetrahydrofolate reductase (MTHFR) gene polymorphism is commonly existent. Research showed that the genetic mutagenesis of MTHFR offers remission of cancer cell formation under sufficient folate status; however, this mutagenesis is in turn a risk factor for some carcinoma under folate deficiency. The present mutation mechanisms have not yet been clear. In this thesis, I aim to disrupt the expression of MTHFR via the lentiviral RNAi system in order to research upon the reciprocal effects between MTHFR and environmental folate status, and to discuss the relationships of MTHFR with human hepatoma cell cycle distribution, folate metabolism and low enzymatic activity of MTHFR and its effects on human hepatoma cell tolerance under environmental folate status. In the systemic model of MTHFR disruption in human hepatoma cells, we found that after MTHFR gene expression is disrupted, its protein and enzymatic activity is subsequently inhibitied. This may cause changes in the HepG2 cell cycle as well as in its ability to metabolize folate, which effects human hepatoma cell tolerance under environmental folate status. This result proves an intimate relationship between MTHFR gene expression and folate status. At the same time, we consider that this regulatory mechanism may be one of the reasons for affecting the cancer process due to MTHFR gene mutation. This thesis addresses the intimate relationships among MTHFR, growth status of human hepatoma cells and environmental folate status. The establishment of this system may provide future reference materials and foundations for cancer therapy.