核糖核酸干擾 (RNAi) 是一種新穎的基因表現調控機制，能抑制轉錄作用或藉由序列專一性RNA降解程序 (後轉錄基因沈默 [PTGS] ) 以限制轉錄產物的表現。基因沈默機制是由小片段干擾RNA (siRNA) 或短髮夾狀RNA (shRNA) 所起動，最終導致含有同源序列的目標mRNA被分解。RNAi能抑制轉位子與病毒基因等移動性遺傳因子的活性，故其功能被認為是幫助穩定細胞基因體與對抗病毒感染。最近，RNAi已經成為最有效也最被廣泛使用的基因調節方法，不僅用以幫助臨床疾病之分子治療，也是逆向功能基因體研究上的重要工具。本論文就是針對RNAi之治療運用與基因體功能研究進行探討。
人類B型肝炎病毒 (HBV) 的表面抗原 (HBsAg) 已經被證實是誘發肝臟疾病的高危險因子，然而HBsAg本身的突變性卻是導致治療困難的主因。許多報告顯示，RNAi技術是極具潛力的基因沈默治療方法，可用於抑制病毒的感染與複製。於此部分研究中，我們探討RNAi對HBV短期或長期感染下的治療潛力。經由詳細的篩選後證實，有效的shRNA很可能成為一種強效性藥物，能針對HBsAg誘發之疾病進行治療。此外，於細胞內同時表現多種siRNA或shRNA就能同時抑制多種基因或對同一基因的不同位點進行抑制。此策略相當適合用於複雜的基因功能網絡與訊息傳遞路徑等方面的研究，並且此策略亦可用於克服病毒基因突變所造成的治療失效，或同時抑制許多基因表現以達到組合式療法的功效。基於上述優點，我們以目前最為廣泛應用於哺乳細胞的pSUPER質體為基礎，設計一套首尾串聯組合式多重shRNA表現系統的構築策略。此策略提供一簡單而迅速的流程以建構多重shRNA表現載體，並快速而精確的鑑定選殖質體的正確性。我們並證明此多重shRNA表現載體不僅能抑制各相對應的六種基因的表現，同時在誘發前列腺癌細胞凋亡上有不同程度的效果。除此之外，此策略還能運用於任何以DNA載體為基礎的shRNA表現系統。
過去發現，惡性腫瘤細胞偏好以無氧糖解作用產生細胞所需能量並降低有養呼吸作用，此現象稱為瓦伯效應，其發生被認為可能與腫瘤細胞內之粒線體功能異常有關。檸檬酸合成酶 (CS) 是參與檸檬酸循環 (TCA cycle) 中的第一個酵素，也是影響反應速率的關鍵酵素。於此部分的研究，我們運用RNAi技術探索CS蛋白進入粒線體的可能機制，同時也試圖證明CS蛋白與腫瘤惡性化的關連性。結果顯示，由粒線體導向序列 (mitochondrial targeting sequence, MTS) 所調控之CS蛋白進入粒線體的機制中，MTS內的R9胺基酸是關鍵殘基 (residue)，而TOM20、TOM22與TOM70等三種粒線體輸入接受器也是必須的要素。令人注意的是，當我們持續以RNAi抑制CS蛋白的表現卻導致明顯的細胞外觀變化，此變化類似於上皮細胞轉形成間葉細胞 (epithelial-mesenchymal transition, EMT) 的現像。經鑑定後發現有上皮細胞標記蛋白E-cadherin表現下降與間葉細胞標記蛋白alpha-SMA表現增加的現像，同時也發現F-actin整合成較長的應力纖維。綜合上述結果我們清楚證明，以RNAi技術持續沈默人類CS蛋白的表現會破壞粒線體的生物能量生產，使細胞轉為糖解代謝，最終誘導子宮頸癌細胞產生EMT現像，而這可能是造成腫瘤細胞轉移的主因。

RNA interference (RNAi) is a novel regulatory mechanism of gene expression that limits the transcript level by suppressing transcription or through a sequence-specific RNA degradation progress (posttranscriptional gene silencing [PTGS]). Gene silencing can be triggered by small interfering RNA (siRNA) or short hairpin RNA (shRNA), and eventually destroy the cognate target mRNA. The nature function of RNAi seem to protect the genome by inhibit the mobile genetic elements and virus infections. Recently, RNAi have become the most powerful and widely used gene silencing approach for molecular therapeutics and reverse functional genomics.
Human hepatitis B virus (HBV) surface antigen (HBsAg) has proven to be a significant risk factor in HBV induced liver diseases, and an increasing number of mutations in HBsAg are known to increase the difficulty in therapeutic interventions. RNAi technologies have been report as an effectively potent method for gene silencing-based therapeutics to inhibit virus infection or replication. In this study, we explore the therapeutic potential of RNAi on HBV induced diseases in particular resulted from transient or persistent expression of HBsAg. Our results indicate that potent shRNA molecules can be considered as a powerful therapeutic agent on HBsAg induced diseases. Co-delivery or co-expression of multiple siRNAs results in either inhibiting multiple genes simultaneously or targeting multiple sites on a single gene at the same time. These approaches can be used for uncovering functional redundancy in gene networks or dissecting complex signal transduction pathways, and applied for counteracting viral escape by mutation variants or targeting multiple components in combination therapy. For these advantages, we have established a simple and efficient strategy for constructing head-to-tail tandem array multiple shRNAs expression system based on the most popular expression vector pSUPER in mammalian cells. This strategy provides a simple and easy way for efficiently constructing the multiple shRNA expression vectors and quickly and precisely mapping the cloned constructs. We had proved the multiple shRNAs expression vectors could not only efficiently inhibit all six genes but also significantly induce apoptosis at different levels. Moreover, this strategy can be directly applied to any other DNA vector-based shRNA expression system.
It have been report that malignly cancer cells preferentially utilize anaerobic glycolysis for energy generation while down-regulating their aerobic respiration (or oxidative phosphorylation) in the mitochondria the so-called “Warburg effect”. Citrate synthase (CS) is the first and rate-limiting enzyme of the tricarboxylic acid (TCA) cycle. In this part of study, RNAi technology was use to explore the translocation mechanism and discover the role of CS in cancer malignant. The results suggest that the R9 residue should be a key residue of the mitochondrial targeting signal (MTS) and all three preprotein import receptors, including TOM20, TOM22 and TOM70, are required for the N-terminal mitochondrial targeting sequence-mediated transporting human CS protein into mitochondria. Remarkably, persistent long-term RNAi-mediated knockdown of CS expression resulted in dramatically morphological change that closely resembles the epithelial-mesenchymal transition (EMT) phenotype, characterized by down-regulation of the epithelial marker E-cadherin and up-regulation of the mesenchymal marker alpha-smooth muscle actin (alpha-SMA), as well as reorganization of the F-actin into long stress fibers. This proof-of-principle study clearly demonstrates that the RNAi-mediated silencing of the human CS expression induces EMT phenotype resulting from disrupting the mitochondrial bioenergetics in turn promoting the glycolytic metabolism that may be one of the important factors for cancer cell metastasis.

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