生成生物能量胞器-粒線體在細胞功能上，在生長、分裂、能量代謝以及細胞凋亡上扮演決定性角色。粒線體生物能的缺失將使細胞轉換到更原始使用細胞質的糖解路徑作為能量的產生以及促進惡性腫瘤。最近，我們已經提出利用核糖核酸干擾技術來持續沉默檸檬酸酸合成酶，檸檬酸循環中第一個酵素以及速率決定步驟，會誘導上皮-間質轉化(EMT)的表現型，並且在人類子宮頸癌細胞株HeLa及SiHa皆會導致腫瘤轉移以及入侵的現象。雖然我們已經提出沉默人類檸檬酸酶的表現會干擾粒線體的生物能路徑並且會促進細胞質糖解的代謝路徑，但是這異常代謝改變的詳細機制並不是完整的解釋。在這次研究中，我們已經顯示出人類子宮頸癌細胞株HeLa中，人類檸檬酸合成酶的沉默會導致上皮-間質轉化(EMT)的表現型特徵和p53的缺失有很大的關連。除此之外，我們也顯示上皮-間質轉化(EMT)的表現型可以透過MG132處理以及沉默MDM2 (HDM2)的表現所回復。另外，我們也顯示抑制人類檸檬酸合成酶在人類乳腺癌細胞(wide type p53)會導致上皮-間質轉化(EMT)的表現型只有在同時沉默人類p53的表現下。在MCF7細胞株中沉默人類檸檬酸合成酶及人類p53會誘導上皮-間質轉化(EMT)的表現型，顯示要誘導上皮-間質轉化(EMT)的表現型只有在人類檸檬酸合成酶及人類p53的表現同時失能下。然而，我們還另外顯示沉默人類檸檬酸合成酶會誘導上皮-間質轉化(EMT)的表現型在人類攝護腺腫瘤細胞(null p53)。這個結論清楚的顯示p53的缺失要去誘導上皮-間質轉化(EMT)的表現型需要同時沉默人類檸檬酸合成酶。而且有學者提出p53可去調控糖解路徑而藉由它所調控的p53 induced glycolysis and apoptosis regulator (TIGAR)以及p53可去促進氧化磷酸化透過synthesis of cytochrome c oxidase 2 (SCO2) 的活化. 。根據此結果，持續沉默人類檸檬酸合成酶的細胞株會展示出強烈的調降TIGAR以及SCO2的表現,由於HDM2所調控的p53所造成透過泛素化所導致p53的降解，而造成細胞生物能量的轉換從粒線體的氧化磷酸化到細胞質糖解的腫瘤生成。

The bioenergetic organelle mitochondrial plays an important roles in a variety of cellular functions including growth, division, energy metabolism and apoptosis. Defects in mitochondrial bioenergetic will led the cell revert to a more primitive cytoplasmic glycolysis for energy generation and promote to tumor malignancy. Recently, we have shown that persistently knockdown the RNAi-mediated silencing of the human citrate synthase (CS), the first and rate-limiting enzyme of the tricarboxylic acid cycle (TCA), dramatically induced epithelial-mesenchymal transition (EMT), resulting in increased tumor migration and invasion in HeLa and SiHa human uterine cervix cancer cells. Although we have provided the concept about knockdown of the human CS expression disrupted the mitochondrial bioenergetic pathway and in turn promoted the cytosolic glycolysis metabolism, but the detailed mechanism of this abnormal metabolic alteration is not completely elucidated yet. In this study, we have first demonstrated that inhibition of the human CS expression induced EMT phenotype in human cervical carcinoma cell line HeLa (impaired p53). Besides, we have shown that this phenotypic switch could be revered by treatment with MG132, an ubiquitin-proteasome inhibitor, or knockdown of the human homolog of MDM2 (HDM2). In addition, we have also demonstrated that silencing of the human CS expression induced EMT phenotype in human breast cancer cell line MCF7 (wild type p53), under the only condition of co-knockdown of the human p53 expression. Silencing of neither the human CS expression nor the human p53 expression could induce EMT phenotype in MCF-7 cells, suggesting that the EMT phenotype was induced only by dysfunction of the both the human CS and p53 expression. Moreover, we have additionally shown that silencing of the human CS expression induced EMT phenotype in human prostate cancer cell line PC3 (null p53) and that this phenotype switch could not be reversed by knockdown of the human HDM2 expression .These results clearly indicate that impairment of p53 is required for induction of the MET phenotype by silencing of the human CS expression. It has been well characterized that the human p53 inhibits glycolysis through p53 induced glycolysis and apoptosis regulator (TIGAR) induction and promotes oxidative phosphorylation (OXPHOS) through synthesis of cytochrome c oxidase 2 (SCO2) activation. According to this effect, the human CS-knockdown cell lines exhibited a strong down-regulator of the TIGAR and SCO2 expression, resulting from rapid HDM2-mediated p53 degradation by ubiquitin-mediated p53 degradation switches the cellular bioenergetic metabolism form mitochondrial OXPHOS to cytosolic glycolysis that as an intrinsic carcinogenesis.

Adelman, Z.N., Sanchez-Vargas, I., Travanty, E.A., Carlson, J.O., Beaty, B.J., Blair, C.D., and Olson, K.E. (2002). RNA silencing of dengue virus type 2 replication in transformed C6/36 mosquito cells transcribing an inverted-repeat RNA derived from the virus genome. J Virol 76, 12925-12933.
Ashrafi, K., Chang, F.Y., Watts, J.L., Fraser, A.G., Kamath, R.S., Ahringer, J., and Ruvkun, G. (2003). Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 421, 268-272.
Batlle, E. et al. (2000). The transcription factor Snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nature Cell Biol. 2, 84–89.

Bauer, D.E., Hatzivassiliou, G., Zhao, F., Andreadis, C., and Thompson, C.B. (2005). ATP citrate lyase is an important component of cell growth and transformation. Oncogene 24, 6314-6322.
Bensaad, K., Tsuruta, A., Selak, M.A., Vidal, M.N.C., Nakano, K., Bartrons, R., Gottlieb, E., and Vousden, K.H. (2006). TIGAR, a p53-Inducible Regulator of Glycolysis and Apoptosis Cell 126, 30-32.

Brahimi-Horn, M.C., and Pouyssegur, J. (2005). The hypoxia-inducible factor and tumor progression along the angiogenic pathway. Int Rev Cytol 242, 157–213.

Brizel, D.M., Schroeder, T., Scher, R.L., Walenta, S., Clough, R.W., Dewhirst, M.W., and Mueller-Klieser, W. (2001). Elevated tumor lactate concentrations predict for an increased risk of metastases in head-and-neck cancer. Int J Radiat Oncol Biol Phys 51, 349-353.
Brummelkamp, T.R., Bernards, R., and Agami, R. (2002b). A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550-553.
Brunner, E.C., Romeike, B.F., Jung, M., Comtesse, N., and Meese, E. (2006). Altered expression of beta-catenin/E-cadherin in meningiomas. Histopathology 49, 178-187.
Brusselmans, K., Vrolix, R., Verhoeven, G., and Swinnen, J.V. (2005). Induction of cancer cell apoptosis by flavonoids is associated with their ability to inhibit fatty acid synthase activity. J Biol Chem 280, 5636-5645.
Bullock, A.N., and Fersht, A.R. (2001). Rescuing the function of mutant p53. Nat Rev Cancer 1, 68-76.
Caplen, N.J., Zheng, Z., Falgout, B., and Morgan, R.A. (2002). Inhibition of viral gene expression and replication in mosquito cells by dsRNA-triggered RNA interference. Mol Ther 6, 243-251.
Chi, J.T., Chang, H.Y., Wang, N.N., Chang, D.S., Dunphy, N., and Brown, P.O. (2003). Genomewide view of gene silencing by small interfering RNAs. Proc Natl Acad Sci U S A 100, 6343-6346.
Chiu, Y.L., and Rana, T.M. (2002). RNAi in human cells: basic structural and functional features of small interfering RNA. Mol Cell 10, 549-561.
Cioca, D.P., Aoki, Y., and Kiyosawa, K. (2003). RNA interference is a functional pathway with therapeutic potential in human myeloid leukemia cell lines. Cancer Gene Ther 10, 125-133.
Clemens, J.C., Worby, C.A., Simonson-Leff, N., Muda, M., Maehama, T., Hemmings, B.A., and Dixon, J.E. (2000). Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc Natl Acad Sci U S A 97, 6499-6503.
Davidson, B.L., and Paulson, H.L. (2004). Molecular medicine for the brain: silencing of disease genes with RNA interference. Lancet Neurol 3, 145-149.
De Fougerolles, A., Vornlocher, H.P., Maraganore, J., and Lieberman, J. (2007). Interfering with disease: a progress report on siRNA-based therapeutics. Nat Rev Drug Discov 6, 443-453.
Depicker, A., and Montagu, M.V. (1997). Post-transcriptional gene silencing in plants. Curr Opin Cell Biol 9, 373-382.
Douglas, B.W., and Karen, H.V. (2001). Regulation of p53 Function. 264, 56-66.
Douglas, R.G., and John, C. R. (1998). Mitochondria and apoptosis. Science 281, 1309-1312.
Dykxhoorn, D.M., Novina, C.D., and Sharp, P.A. (2003). Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol 4, 457-467.
Elstrom, R.L., Bauer, D.E., Buzzai, M., Karnauskas, R., Harris, M.H., and Plas, D.R. (2004). Akt stimulates aerobic glycolysis in cancer cells. Cancer Res 64 , 3892–3899.
Fantin, V.R., St-Pierre, J., and Leder, P. (2006). Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance. Cancer Cell 9, 425-434.
Filleur, S., Courtin, A., Ait-Si-Ali, S., Guglielmi, J., Merle, C., Harel-Bellan, A., Clezardin, P., and Cabon, F. (2003). SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth. Cancer Res 63, 3919-3922.
Flier, J.S., Mueckler, M.M., Usher, P., and Lodish, H.F. (1987). Elevated Levels of Glucose Transport and Transporter Messenger RNA are Induced by ras or src Oncogenes. Science 235, 1492–1495.

Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811.
Fraser, A.G., Kamath, R.S., Zipperlen, P., Martinez-Campos, M., Sohrmann, M., and Ahringer, J. (2000). Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408, 325-330.
Hamilton, A.J., and Baulcombe, D.C. (1999). A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950-952.
Hammond, S.M., Bernstein, E., Beach, D., and Hannon, G.J. (2000). An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404, 293-296.
Harper, S.Q., Staber, P.D., He, X., Eliason, S.L., Martins, I.H., Mao, Q., Yang, L., Kotin, R.M., Paulson, H.L., and Davidson, B.L. (2005). RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model. Proc Natl Acad Sci U S A 102, 5820-5825.
McCaffrey, A.P., Nakai, H., Pandey, K., Huang, Z., Salazar, F.H., Xu, H., Wieland, S.F., Marion, P.L., and Kay, M.A. (2003). Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol 21, 639-644.
Napoli, C., Lemieux, C., and Jorgensen, R. (1990). Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell 2, 279-289.
Gitlin, L., and Andino, R. (2003). Nucleic acid-based immune system: the antiviral potential of mammalian RNA silencing. J Virol 77, 7159-7165.
Gitlin, L., Stone, J.K., and Andino, R. (2005). Poliovirus escape from RNA interference: short interfering RNA-target recognition and implications for therapeutic approaches. J Virol 79, 1027-1035.
Gonczy, P., Echeverri, C., Oegema, K., Coulson, A., Jones, S.J., Copley, R.R., Duperon, J., Oegema, J., Brehm, M., Cassin, E., et al. (2000). Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 408, 331-336.
Hatzivassiliou, G., Zhao, F., Bauer, D.E., Andreadis, C., Shaw, A.N., Dhanak, D., Hingorani, S.R., Tuveson, D.A., and Thompson, C.B. (2005b). ATP citrate lyase inhibition can suppress tumor cell growth. Cancer Cell 8, 311-321.
Hay, E.D. (1995). An overview of epithelio-mesenchymal transformation. Acta
Anat. (Basel) 154, 8–20.

Hay, E.D. (2005). The mesenchymal cell, its role in the embryo, and the remarkable signaling mechanisms that create it. Dev. Dyn. 233, 706–720.

Hung, C.F., Lu, K.C., Cheng, T.L., Wu, R.H., Huang, L.Y., Teng, C.F., and Chang, W.T. (2006). A novel siRNA validation system for functional screening and identification of effective RNAi probes in mammalian cells. Biochem Biophys Res Commun 346, 707-720.
Jacque, J.M., Triques, K., and Stevenson, M. (2002). Modulation of HIV-1 replication by RNA interference. Nature 418, 435-438.
Jiang, M., and Milner, J. (2003). Bcl-2 constitutively suppresses p53-dependent apoptosis in colorectal cancer cells. Genes Dev 17, 832-837.
Kapadia, S.B., Brideau-Andersen, A., and Chisari, F.V. (2003). Interference of hepatitis C virus RNA replication by short interfering RNAs. Proc Natl Acad Sci U S A 100, 2014-2018.
Kittler, R., and Buchholz, F. (2003). RNA interference: gene silencing in the fast lane. Semin Cancer Biol 13, 259-265.
Krebs, H.A. (1940). The citric acid cycle: A reply to the criticisms of F. L. Breusch and of J. Thomas. Biochem J 34, 460-463.
Lu, P.Y., Xie, F.Y., and Woodle, M.C. (2005). Modulation of angiogenesis with siRNA inhibitors for novel therapeutics. Trends Mol Med 11, 104-113.
Martinez-Zaguilan, R., Seftor, E.A., Seftor, R.E., Chu, Y.W., Gillies, R.J., and Hendrix, M.J. (1996). Acidic pH enhances the invasive behavior of human melanoma cells. Clin Exp Metastasis 14, 176-186.
Paddison, P.J., Caudy, A.A., Bernstein, E., Hannon, G.J., and Conklin, D.S. (2002). Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 16, 948-958.
Paul, C.P., Good, P.D., Winer, I., and Engelke, D.R. (2002). Effective expression of small interfering RNA in human cells. Nat Biotechnol 20, 505-508.
Pelicano, H., Martin, D.S. , and Huang, P. (2006). Glycolysis inhibition for anticancer treatment. Oncogene 25, 4633–4646.
Rempel, A., Mathupala, S.P., Griffin, C.A., Hawkins, A.L., and Pedersen,
P.L. (1996). Aberrant glycolytic metabolism of cancer cells: a remarkable coordination of genetic, transcriptional, post-translational, and mutational events that lead to a critical role for type II hexokinase. Cancer Res, 562468–562471.

Ristow, M., Pfister, M.F., Yee, A.J., Schubert, M., Michael, L., Zhang, C.Y., Ueki, K., Michael, M.D., 2nd, Lowell, B.B., and Kahn, C.R. (2000). Frataxin activates mitochondrial energy conversion and oxidative phosphorylation. Proc Natl Acad Sci U S A 97, 12239-12243.
Rozhin, J., Sameni, M., Ziegler, G., and Sloane, B.F. (1994). Pericellular pH affects distribution and secretion of cathepsin B in malignant cells. Cancer Res 54, 6517-6525.
Satoaki M., Ju-Gyeong K., Willmar D.P., Andrew W., Manfred B., Oksana G., Paula J. H., Fred B., and Paul M. H. (2006). p53 Regulates Mitochondrial Respiration. Science 312, 1650-1653.
Schulz, T.J., Thierbach, R., Voigt, A., Drewes, G., Mietzner, B., Steinberg, P., Pfeiffer, A.F., and Ristow, M. (2006). Induction of oxidative metabolism by mitochondrial frataxin inhibits cancer growth: Otto Warburg revisited. J Biol Chem 281, 977-981.
Schwickert, G., Walenta, S., Sundfor, K., Rofstad, E.K., and Mueller-Klieser, W. (1995). Correlation of high lactate levels in human cervical cancer with incidence of metastasis. Cancer Res 55, 4757-4759.
Selak, M.A., Armour, S.M., MacKenzie, E.D., Boulahbel, H., Watson, D.G., and Mansfield K.D. et al. (2005). Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-α prolyl hydroxylase . Cancer Cell 7, 77–85.

Serkova, N., and Boros, L.G. (2005). Detection of resistance to imatinib by metabolic profiling: clinical and drug development implications. Am J Pharmacogenomics 5, 293–302.

Shlomai, A., and Shaul, Y. (2003). Inhibition of hepatitis B virus expression and replication by RNA interference. Hepatology 37, 764-770.
Shuey, D.J., McCallus, D.E., and Giordano, T. (2002). RNAi: gene-silencing in therapeutic intervention. Drug Discov Today 7, 1040-1046.
Stark, G.R., Kerr, I.M., Williams, B.R., Silverman, R.H., and Schreiber, R.D. (1998).
Stubbs, M., McSheehy, P.M., Griffiths, J.R., and Bashford, C.L. (2000). Causes and consequences of tumour acidity and implications for treatment. Mol Med Today 6, 15-19.
Tayeau, F., and Neuzil, E. (1951). Krebs' cycle and the physiopathology of its process. J Med Bord 128, 865-880.
Taylor, R.W., Turnbull, D.M. (2005). Mitochondrial DNA mutations in human disease. Nat Rev Genet 6, 389–402.
Thiery, J.P., Epithelial-mesenchymal transitions in tumour progression. Nature Rev.
Cancer 2, 442–454 (2002).

Thiery, J.P. , and Sleeman, J. P. (2006). Complex networks orchestrate epithelial-mesenchymal transitions. Nature Rev. Mol. Cell Biol. 7, 131–142.

Thompson, E.W., Newgreen, D. F. and Tarin, D. (2005). Carcinoma invasion and metastasis: a role for epithelial–mesenchymal transition? Cancer Res. 65, 5991–5995.

Tuschl, T. (2003). Functional genomics: RNA sets the standard. Nature 421, 220-221.
Vaupel, P., Okunieff, P., and Neuringer, L.J. (1989). Blood flow, tissue oxygenation, pH distribution, and energy metabolism of murine mammary adenocarcinomas during growth. Adv Exp Med Biol 248, 835-845.
Vogelstein, B., and Kinzler, K.W. (1992). p53 function and dysfunction. Cell 21, 523-526.
Walenta, S., and Mueller-Klieser, W.F. (2004). Lactate: mirror and motor of tumor malignancy. Semin Radiat Oncol 14, 267-274.
Walenta, S., Salameh, A., Lyng, H., Evensen, J.F., Mitze, M., Rofstad, E.K., and Mueller-Klieser, W. (1997). Correlation of high lactate levels in head and neck tumors with incidence of metastasis. Am J Pathol 150, 409-415.
Walenta, S., Wetterling, M., Lehrke, M., Schwickert, G., Sundfor, K., Rofstad, E.K., and Mueller-Klieser, W. (2000). High lactate levels predict likelihood of metastases, tumor recurrence, and restricted patient survival in human cervical cancers. Cancer Res 60, 916-921.
Warburg, O. (1956). On the origin of cancer cells. Science 123, 309-314.
William, M. and Miklo´s, M. (1998). The hydrogen hypothesis for the first eukaryote. Nature 392, 37-41.

Wilson, J.A., Jayasena, S., Khvorova, A., Sabatinos, S., Rodrigue-Gervais, I.G., Arya, S., Sarangi, F., Harris-Brandts, M., Beaulieu, S., and Richardson, C.D. (2003). RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. Proc Natl Acad Sci U S A 100, 2783-2788.
Wu, M.T., Wu, R.H., Hung, C.F., Cheng, T.L., Tsai, W.H., and Chang, W.T. (2005b). Simple and efficient DNA vector-based RNAi systems in mammalian cells. Biochem Biophys Res Commun 330, 53-59.
Yang, J., Mani, S.A., and Weinberg, R.A. (2006). Exploring a new twist on tumor
metastasis. Cancer Res. 66, 4549–4552.

Zamore, P.D., Tuschl, T., Sharp, P.A., and Bartel, D.P. (2000). RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25-33.