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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/8360

Title: 粒線體功能缺損導致人類肝癌細胞惡化---VLDLR與脂質代謝之角色
Mitochondrial Dysfunction-Induced Cancer Progression in Human Hepatoma Cells---The Roles of Vldlr and Lipid Metabolism
Authors: 張君如
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 化療抗性(chemo-resistance);遷移能力(migration);侵襲能力(invasion);總膽固醇(total-cholesterol);三酸甘油酯(triglyceride);粒線體逆行訊息(mitochondrial retrograde signaling)
chemo-resistance;migration;invasion;total-choloesterol;triglyceride;and mitochondrial retrograde signaling
Date: 2010-08
Issue Date: 2011-06-28T05:50:59Z
Publisher: 行政院國家科學委員會
Abstract: 摘要:粒線體功能缺損導致人類肝癌細胞惡化:VLDLR與脂質代謝之角色 背景:代謝反應改變是癌細胞惡化過程的現象之一。人類癌組織常見粒線體DNA變異及呼吸鏈酵素功能失常,近年我們在人類肝癌細胞株研究發現:粒線體功能缺損誘發化療抗性及細胞遷移等癌惡化特徵。其中,粒線體功能缺損所引發細胞內鈣離子移動及活性氧分子(ROS)增加,可能調控細胞核基因表現及生物功能。然而,人類肝癌細胞粒線體功能缺損如何改變代謝反應、影響癌細胞惡化特徵仍是亟待探究的問題。因此,我們先根據粒線體功能缺損時基因表現量變化,預測其參與之生物反應,結果顯示:脂質、脂肪酸及膽固醇代謝等均可能受其調控。初步以即時定量聚合酶連鎖反應分析發現:對人類肝癌細胞株HepG2及HA22T/VGH,粒線體功能缺損造成極低密度脂蛋白受體(VLDLR)過度表現。VLDLR主要功能為促進細胞脂質代謝及運輸作用、但其對癌細胞惡化之影響仍不明確。本研究假設:人類肝癌細胞粒線體功能缺損時,粒線體調控訊息分子造成VLDLR過表現,提高脂質代謝、改變癌細胞惡化。 方法:模式一:HepG2轉染siRNA抑制粒線體DNA轉錄或給予粒線體呼吸鏈酵素抑制劑,分別造成粒線體基因缺損或呼吸功能缺損,評估細胞耗氧率、ATP消耗、細胞內鈣離子移動及ROS生成。以即時定量聚合酶連鎖反應及西方點墨法定量VLDLR基因表現,以VLDLR siRNA或專一抗體評估粒線體缺損時VLDLR對脂質代謝、細胞增殖、轉移及化療抗性的影響(分別定量細胞內總膽固醇及三酸甘油酯、進行cell growth assay、wound healing assay、transwell migration assay、transwell invasion assay、LDH assay、cell cycle analysis)。模式二:HepG2轉染載體造成VLDLR過表現,評估VLDLR對HepG2脂質代謝、細胞增殖、轉移及化療抗性(分析法如模式一)。製作一系列VLDLR啟動子PPRE、C/EBP或NF-Y調控區刪除變異的pGL3冷光酵素報告質體,分析粒線體缺損引發之訊息分子(鈣離子、ROS…)對VLDLR基因表現及脂質代謝的影響;並以此系列報告質體篩選食物、植物及海洋來源活性成分,評估該成分對粒線體缺損時癌細胞惡化特徵之影響。 預期成果:第一年,人類肝癌細胞株粒線體功能缺損誘發VLDLR基因表現、改變脂質代謝、細胞增殖、化療抗性、遷移及侵襲。第二年,VLDLR過表現對人類肝癌細胞株脂質代謝及癌惡化特徵之影響。第三年,粒線體逆行訊息調控VLDLR基因啓動子轉錄因子及脂質代謝之機制;並呈現食物等來源活性物質對粒線體逆行訊息及癌惡化特徵之作用機轉。綜合而言,本研究將呈現人類肝癌細胞粒線體缺損時VLDLR及脂質代謝之角色、及其調控癌症惡化之機轉,期望作為癌症治療策略參考。
Abstract:
Background: Metabolic reprogramming is one of the phenomena in the cancer progression. Alteration of mitochondrial DNA and malfunction of mitochondrial respiratory chain enzymes have been demonstrated in human cancers. Our recently studies revealed that mitochondrial dysfunction-induced cell migration and chemo-resistance in human hepatoma cells. Meanwhile, the mitochondrial dysfunction induced cytosolic Ca2+ mobilization, and reactive oxygen species (ROS) overproduction may contribute to the changes of nuclear genes expression and biological process. However, the mechanisms of mitochondrial dysfunction-induced metabolic reprogramming and its responsible to cancer progression in hepatoma cells have yet to be elucidated. Therefore, we were investigated the mitochondrial dysfunction-induced differentially expressed genes and annotated their biological functions. We found that the metabolisms of lipid, fatty acid and cholesterol may affected by mitochondrial dysfunction. By using of real-time quantitative PCR, we revealed that mitochondrial dysfunction-induced the overexpression of very low density lipoprotein receptor (VLDLR) in human hepatoma HepG2 and HA22T/VGH cells. VLDLR majorly function in lipid metabolism and transport. However, the evidence of VLDLR in cancer malignance is limited. Together, we have hypothesized that VLDLR could be one of the important target genes involved in the mitochondrial retrograde signaling and to modulate cancer progression through VLDLR and lipid metabolism. Methods: Model 1-- The siRNA of mitochondrial transcription factor and inhibitors of mitochondrial respiratory enzymes will be used to induce mitochondrial genetic stress and respiratory defect in human hepatoma cells, respectively. The mitochondrial dysfunction-induced oxygen consumption, ATP expense, cytosolic Ca2+ mobilization, and ROS production will be measured. The gene expression of VLDLR will be analyzed by real-time quantitative PCR and Western blot, respectively. The siRNA and neutralizing antibody of VLDLR will be used to evaluate its roles in lipid metabolism and cancer malignance. Model 2-- The VLDLR expression vector will be constructed and transfected to HepG2. Then, the lipid metabolism and cancer maligance assays with be analyzed. Additionally, the pGL3 luciferase reporter vectors containing the human VLDLR wildtype and its mutant promoters (ΔPPRE, ΔC/EBP, and ΔNF-Y) will be constructed, and to investigate the transcription factors in response to mitochondrial dysfunction-induced signaling molecules (such as calcium, and ROS, etc.) in human hepatoma cells. Moreover, these constructs will be use to study the effects of marine and botanic bioactive components on cancer malignance. In summary, the results of this study will provide important information of the roles of VLDLR play in the mitochondrial dysfunction-modulated lipid metabolism and cancer malignance in human hepatoma. Further characterization of the lipid metabolism confer with VLDLR induction in human hepatoma is crucial to characterize the potential benefits of combination therapy in the management of this disease.
Relation: NSC99-2320-B019-006-MY3
URI: http://ntour.ntou.edu.tw/ir/handle/987654321/8360
Appears in Collections:[食品科學系] 研究計畫

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