蛋白質是生物體內非常重要的產物，在人體中龐大的新陳代謝機制都是依靠不同蛋白質而產生出不同的功能而形成的一個巨大機制網，然而也不是所有蛋白質都有正常的功能，假如蛋白質遇到受損的或是異常摺疊不完全時，身體內就會有一套非常重要的機制來將之分解，那個最重要的小分子就是泛素。
泛素（ubiquitin）是一種存在於真核細胞的小蛋白，是高度保存的已知蛋白質之一，它的主要功能為標記需要分解掉的蛋白質使其被水解。我們知道在惡性腫瘤的生長或是爬行能力上面都比其他細胞來得迅速，這代表其代謝的能力及速度勢必也需要加快的，就過去的文獻研究發現，在惡性腫瘤中，泛素化路徑被高度活化的表現是有明顯上升的，因此我們利用RNA干擾技術（RNA interference）來針對惡性腫瘤中的泛素表現量進行穩定抑制，建立穩定默化惡性腫瘤的細胞株來進行以下的研究。
我先從網路資料庫（Human Protein Atlas）中進行搜尋，分析比對泛素在正常組織及腫瘤細胞的表現量，發現無論在正常組織中或是腫瘤細胞都有看到泛素高表現的情形；接著我利用四株不同腫瘤細胞進行RNAi的轉染以建立挑選缺乏泛素表現得穩定默化細胞株，利用拍照觀察、MTT細胞增殖分析、細胞群落分析、傷口癒合實驗、Boyden chamber、細胞週期分析等實驗中都發現在缺乏泛素表現的細胞株相對於對照組其爬行能力以及細胞生長能力都有顯著的降低。
為了更進一步了解在缺乏泛素的情形下對於藥物的敏感性，因此我們利用了三種不同的抗癌藥物二去氧葡萄糖、每福敏、依托泊苷（2-Deoxy-D-glucose、Metformin、Etoposide）來偵測其細胞的變化，我們發現在二去氧葡萄糖的處理下，缺乏泛素表現的細胞株相對於對照組都有更明顯降低細胞生長能力以及毒殺能力，顯示出穩定默化泛素細胞株有著較高的藥物敏感性，並且隨著不同濃度的藥物處理會有不同的殺傷效果；而在每福敏的處理下，也有藥物敏感性及Dosage effect，但是效果相較於2-DG比較起來是比較弱的；最後在依托泊苷的處理中，由於依托泊苷的殺傷力很強，直接針對topoisomerase II進行攻擊，因此我們在泛素默化細胞株以及對照組是沒有發現明顯的差異。最後我們也在西方點墨法（Western blot）的分析發現細胞週期的相關蛋白表現量降低，這可能來說明造成細胞生長變慢的一項的主要原因。
歸納以上結果，我們可以發現在惡性腫瘤中缺乏泛素的情形下可以使得腫瘤細胞的惡性程度降低，這些都是未來在治療腫瘤上面的一項新指標。

Ubiquitin is a small protein that has involved in almost all eukaryotic organisms and it is highly conserved. It consists of 76 amino acids and has a molecular mass of about 8.5 kDa. The function of ubiquitin is to specifically target the proteins that need to be degraded. Recent studies show that many types of cancer present the high level of ubiquitin, which promotes cancer cell migration and tumor growth as compared with the normal cell. To approach more detail results, I have established a stable knockdown ubiquitin in four different malignant cancer cells, including HeLa, H1299, MDA-MB-231 and PC3 by applying the RNAi technique. Analysis of the knockdown cell line shows that loss of ubiquitin expression would decreased the cell growth, migration, colony formation ability and colony numbers. Moreover, to understand the drug sensitivity between the stable knockdown cell lines and control cell lines, I use 2-Deoxy-D-glucose, metformin and etoposide to examine the difference. The result shows that stable knockdown cell lines have a higher drug sensitivity and have the dosage effect. Beside these results, flow cytomertry assays show that loss of ubiquitin casues the change of cell cycle distribution and a higher amount of G2/M phase. Furthermore, western blot assays also demonstrate that many cyclin proteins such as cdc2 and cyclinB1 are downregulated. Taken together, the ubiquitin deficiency on malignant cancer cell may be potential anti-tumor treatment in the future.

[1] William, A., Dunn, J. Autophagy and related mechanisms of lysosome-mediated
protein degradation. Cell Press. 139–143 (1994).
[2] J F Dice. Molecular determinants of protein half-lives in eukaryotic cells. The
FASEB Journal 5, 349-357(1987).
[3] Goldstein, G., et al. Isolation of a polypeptide that has lymphocyte
differentiating properties and is probably represented universally in living cells.
Proc Natl Acad Sci. USA 72, 11–15(1975).
[4] Aaron Ciechanover. The ubiquitin-proteasome proteolytic pathway. Cell Press
7,13-21(1994).
[5] Choongseob, O., Soonyong, P., et al. Downregulation of ubiquitin level via
knockdown of polyubiquitin gene Ubb as potential cancer therapeutic
intervention. Scientific Reports 3. (2013) .
[6] Mark Hochstrasser. Evolution and function of ubiquitin-like protein-conjugation
systems. Nature Cell Biology 2, 153 – 157(2000).
[7] Simone, F., Krishnaraj, R., et al. Ubiquitylation in immune disorders and
cancer：from molecular mechanisms to therapeutic implications. EMBO
Molecular Medicine 4, 545-556(2012).
[8] Jiahong, L., Liqiang, H., et al. NRBF2 regulates autophagy and prevents liver
injury by modulating Atg14L-linked phosphatidylinositol-3 kinase III activity.
Nature Communications, 5,(2014)

[9] S. S. Wing, A. L. Goldberg. Glucocorticoids activate the
ATP-ubiquitin-dependent proteolytic system in skeletal muscle during fasting.
American Journal of Physiology.(1993).
[10] Maurizio Bossola, Maurizio Muscaritoli. Increased muscle ubiquitin mRNA
levels in gastric cancer patients. American Journal of Physiology. .
1518-1523.(2001).
[11] J.-L. Qu1, X.-J. Qu. Gastric cancer exosomes promote tumour cell proliferation
through PI3K/Akt and MAPK/ERK activation.(2009).
[12] Irfan Rahman, William MacNee. Role of transcription factors in inflammatory
lung diseases. Thorax ,601-612(1998).
[13] Ciechanover, A., Daniel, D., et al. Ubiquitin dependence of selective protein
degradation demonstrated in the mammalian cell cycle mutant ts85. Cell ,
37,57-66(1984).
[15] Rodrigues, N., Rowan, A., et al. p53 mutations in colorectal cancer. Proc Natl
Acad Sci 19,7555-7559(1990).
[16] Kaelin, W. The von Hippel-Lindau tumor suppressor gene and kidney cancer.
Clin Cancer Res ,10,(2004)
[17] Battagli, C., Uzzo, R.,et al. Promoter hypermethylation of tumor suppressor
genes in urine from kidney cancer patients. Cancer Res. 24,8695-8699(2003).
[18] Cowey, C., Kimryn,R., et al. VHL Gene Mutations in Renal Cell Carcinoma:
Role as a Biomarker of Disease Outcome and Drug Efficacy. Curr Oncol Rep.
2 ,94-101(2010).

[19] Gnarra, J., et al. Mutations of the VHL tumour suppressor gene in renal
carcinoma. Nature Genetics 7, 85 - 90 (1994).
[20] Jiang,Y., et al. Gene Expression Profiling in a Renal Cell Carcinoma Cell Line
Dissecting VHL and Hypoxia-Dependent Pathways. Mol Cancer Res 1, 453–
462(2003).
[21] Yelena, Y., et al. Non-canonical ubiquitin based signals for proteasomal
degradation. Cell Science. 539-548(2012).
[22] Joerg, H, Lilach, O., et al. Ubiquitin and Ubiquitin-Like Proteins in Protein
Regulation. Circulation Research. Circ Res,1276-1291(2007).
[23] Ciechanover, C., et al. The ubiquitin-proteasome pathway: The complexity and
myriad functions of proteins death. PNAS. 2727–2730(1998).
[24] Hwan-Ching, T., Erin M. Schuman. Ubiquitin, the proteasome and protein
degradation in neuronal function and dysfunction. Nature Reviews
Neuroscience, 9, 826-838(2008).
[25] Mani, A and Gelmann, E. The Ubiquitin-Proteasome Pathway and Its Role
in Cancer. Journal of clinical oncology,23,4776-4789(2005).
[26] Simona,P., Sigismund,S., et al. A single motif responsible for ubiquitin
Recognition and monoubiquitination in endocytic proteins. Nature 416,
451-455(2002).
[27] Sadowski, M., Suryadinata, R., et al. Protein Mono-ubiquitination
and Poly-ubiquitination Generate Structural Diversity to Control Distinct
Biological Processes. IUBMB Life, 64,136–142(2012).

[28] S.Y. Huen, M., et al. BRCA1 and its toolbox for the maintenance of genome
integrity. Nature Reviews Molecular Cell Biology 11, 138-148(2010).
[29] Bremm, A., M V Freund, S., et al. Lys11-linked ubiquitin chains adopt
compact conformations and are preferentially hydrolyzed by the deubiquitinase
Cezanne. Nature Structural & Molecular Biology 17, 939–947 (2010).
[30] Randow, F., Lehner, P. Viral avoidance and exploitation of the ubiquitin
system. Nature Cell Biology 11, 527 - 534 (2009).
[31] Bremm, A., Komander, D. Emerging roles for Lys11-linked polyubiquitin in
cellular regulation. Trends Biochem Sci 7,355-363(2011).
[32] Birsa, N., Norkett, R., et al. Lysine 27 Ubiquitination of the Mitochondrial
Transport Protein Miro Is Dependent on Serine 65 of the Parkin Ubiquitin
Ligase. Journal of Biological Chemistry, 289, 14569-14582 (2014).
[33] Mark Hochstrasser. Ubiquitin, proteasomes, and the regulation of intracellular
protein degradation. Current Opinion in Cell Biology, 7, 215-223(1995).
[34] Zhijian J. Chen. Ubiquitin signalling in the NF-kappaB pathway. Nature Cell
Biology 7, 758 - 765 (2005).
[35] Duncan, L., et al. Lysine‐63‐linked ubiquitination is required for
endolysosomal degradation of class I molecules. The EMBO Journal, 25,
1635-1645(2006).
[36] Bosch, F., Manos, M., et al. Prevalence of Human Papillomavirus in Cervical
Cancer: a Worldwide Perspective. JNCI J Natl Cancer Ins, 11, 796-802(1995).
[37] Jan M. M. Walboomers ., et al. Human papillomavirus is a necessary cause of
invasive cervical cancer worldwide. The Journal of Pathology, 1 ,12-19(1999).
[38] Boscha, F ., et al. The viral etiology of cervical cancer. Elsevier. 183-190.
(2002).
[39] Perou, C., Sørlie, T., et al. Molecular portraits of human breast tumours.
Nature , 406, 747-752(2000).
[40] Lengyel, E., Prechtel, D., et al. c-Met overexpression in node-positive breast
cancer identifies patients with poor clinical outcome independent of Her2/neu.
Journal of Cancer , 113, 678 – 682 (2005).
[41] Skobe, M., Hawighorst, T., et al. Induction of tumor lymphangiogenesis by
VEGF-C promotes breast cancer metastasis. Nature Medicine, 7, 192 - 198
(2001)
[42] Thor, A., Moore II, D., et al. Accumulation of p53 Tumor Suppressor Gene
Protein: An Independent Marker of Prognosis in Breast Cancers. JNCI J Natl
Cancer Inst ,11 , 845-855(1992).
[43] Slamon, D., Shak, S., et al. Use of Chemotherapy plus a Monoclonal Antibody
against HER2 for Metastatic Breast Cancer That Overexpresses HER2. The
New England Journal of Medicine , 344,783-792(2001).
[44] Partin AW, Yoo J. The use of prostate specific antigen, clinical stage and
Gleason score to predict pathological stage in men with localized prostate
cancer. The Journal of Urology, 150(1):110-114(1993).
[45] Zatloukala, P., Petruzelka, L., et al. Concurrent versus sequential
chemoradiotherapy with cisplatin and vinorelbine in locally advanced
non-small cell lung cancer: a randomized study. Elsevier 87–98(2004).

[46] H. Dorota Halicka, Barbara Ardelt. 2-Deoxy-D-Glucose Enhances Sensitivity
of Human Histiocytic Lymphoma U937 Cells to Apoptosis Induced by Tumor
Necrosis Factor. Cancer Res, 55; 44(1995).
[47] Issam Ben Sahra. The combination of metformin and 2-deoxyglucose
inhibits autophagy and induces AMPK-dependent apoptosis in prostate
cancer cells. Autophagy 6:5, 670-671(2010).
[48] Zhou, G., Myers, R., et al. Role of AMP-activated protein kinase in
mechanism of metformin action. J Clin Invest., 108, 1167-1174(2001).
[49] Isaam Ben Sahra, Claire Regazzetti. Metformin, Independent of AMPK,
Induces mTOR Inhibition and Cell-Cycle Arrest through REDD10. AACR ,
3,4366-4372.(2011).
[50] S G Arbuck, H O Douglass. Etoposide pharmacokinetics in patients with
normal and abnormal organ function. JCO , 1690-1695(1986).
[51] Drissi, R., Dubois, M L., et al. Quantitative proteomics reveals dynamic
interactions of the MCM complex in the cellular response to etoposide
induced DNA damage. Molecular & Cellular proteomics ,14, 2002-2013
(2015).
[52] Yao, T., Cohen, R., et al. A cryptic protease couples deubiquitination and
degradation by the proteasome. Nature ,419, 403-407(2002).
[53] Nanduri, B., Suvarnapunya AE., et al. Deubiquitinating enzymes as
promising drug targets for infectious diseases. Curr Pharm Des.
19(18),3234-3247(2013).

[54] Yang, Y., Kitagaki, J., et al. Inhibitors of Ubiquitin-Activating Enzyme (E1),
a New Class of Potential Cancer Therapeutics. Cancer Res, 67 ,
9472-9481(2007).
[55] Huang , H, Ceccarelli , DF., et al. E2 enzyme inhibition by stabilization of a
low-affinity interface with ubiquitin. Nat Chem Biol. 10(2),156-163(2014).
[56] Ambrus Toth, Philip Nickson. Differential Regulation of Cardiomyocyte
Survival and Hypertrophy by MDM2, an E3 Ubiquitin Ligase. The Journal of
Biological Chemistry, 281, 3679-3689(2005)
[57] Ute M. Moll, Oleksi Petrenko. The MDM2-p53 Interaction. Mol Cancer
Res ,1;1001-1008(2003).
[58] Robert C. Kane, Peter F. Bross. Velcade®: U.S. FDA Approval for the
Treatment of Multiple Myeloma Progressing on Prior Therapy. The
Oncologist. 508-513(2003)
[59] Richardson, P., Briemberg, H., et al. Frequency, Characteristics, and
Reversibility of Peripheral Neuropathy During Treatment of Advanced
Multiple Myeloma With Bortezomib. JCO , 19 , 3113-3120 (2006).
[60] Richardson, P., Sonneveld, P., et al. Bortezomib or High-Dose
Dexamethasone for Relapsed Multiple Myeloma. N Engl J
Med ,352,2487-2498(2005).
[61] Bridget N. Fahy. Targeting BCL-2 overexpression in various human
malignancies through NF-κB inhibition by the proteasome inhibitor
bortezomib. Cancer Chemotherapy and Pharmacology. 46-54(2005).