Molecular applications and technological exploitation of RNA interference

• Main Authors: Tsung-lin Cheng, 鄭琮霖
• Other Authors: Wen-tsan Chang
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/46669262897658691182

博士 === 國立成功大學 === 基礎醫學研究所 === 95 === 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.