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系統識別號 U0026-0808201314560100
論文名稱(中文) 人類微核醣核酸-181b(hsa-miR-181b)在大腸直腸癌中所扮演的功能性角色
論文名稱(英文) The functional role of hsa-miR-181b in colorectal cancer
校院名稱 成功大學
系所名稱(中) 生物資訊與訊息傳遞研究所
系所名稱(英) Insitute of Bioinformatics and Biosignal Transduction
學年度 101
學期 2
出版年 102
研究生(中文) 謝婉貞
研究生(英文) Wan-Chen Hsieh
學號 Z26004032
學位類別 碩士
語文別 英文
論文頁數 59頁
口試委員 指導教授-曾大千
口試委員-洪良宜
口試委員-陳百昇
中文關鍵字 大腸直腸癌  微小核糖核酸-181b  5-FU  抗藥性 
英文關鍵字 colorectal cancer  miR-181b  5-FU  drug resistance 
學科別分類
中文摘要 大腸直腸癌在台灣的致死率是第二高的,而其在全世界的死亡率每年也有高達五十萬人之多。關於大腸直腸癌的研究,在臨床上對於病患的預後與治療仍然是相當薄弱。近年來有許多研究指出,許多微小核糖核酸(micro-RNAs)都被發現有參與在大腸直腸癌的發育過程中。在過去文獻中提到微小核糖核酸-181b (miR-181b)在大腸直腸癌中都有較高的表現量,顯示miR-181b可能可以提供一個好的治療指標。在本篇研究中,我們分析了大腸直腸癌病患檢體。萃取檢體中的所有的核糖核酸,利用及時定量聚合酶連鎖反應分析微小核糖核酸及一般的訊息核糖核酸基因。我們確認了miR-181b在腫瘤組織中的表現量都有明顯高於在正常組織中的表現。因此我們進一步利用TargetScan的資料庫找到了微小核糖核酸-181b可能會結合的目標基因,組織金屬蛋白酶抑制物-3 (TIMP-3)。令我們興奮的是,在大腸直腸癌組織與細胞株中,miR-181b與TIMP-3的表現量呈現完全相反的現象。接著我們利用reporter assay發現miR-181b會藉由結合到TIMP-3的3’-UTR而降低TIMP-3的表現量。另外我們在細胞增生以及凋亡實驗的功能性分析中發現了在化療藥物 (5-FU)的處理下,miR-181b對於癌細胞的增生過程似乎會透過調控TIMP-3而在其中扮演重要的角色。首先我們發現擁有比較高miR-181b 表現量的SW620細胞株,對化療藥物 (5-FU)有較高的抗性。更重要的是,我們也發現TIMP-3 會降低原本受miR-181b 誘導增加的細胞增生能力。因此我們推測大腸直腸癌細胞中,可能會透過miR-181b 去降低TIMP-3 的表現而造成細胞對化療藥物變得更具抗藥性。最後我們提出一個理論,假使大腸直腸癌細胞帶有較高表現量的miR-181b和較低表現量的TIMP-3,則此細胞可能偏向屬於抗藥性的細胞株;相反則是對化療藥物較有效。因此我們認為,在大腸直腸癌的預後上,miR-181b與TIMP-3 可以做為治療性的指標。
英文摘要 Colorectal cancer is the second leading cause of death among cancer in Taiwan. It has been estimated that colorectal cancer is responsible for 500,000 deaths worldwide. Progress in diagnosis and treatment for patients with advanced disease or systemic metastasis is still very poor. Several recent reports suggest that microRNAs involved in the development of colorectal cancer. In our studies, the recurrent and non-recurrent colorectal cancer tissues, sixteen and thirty pairs of tumor and normal samples respectively, were analyzed. Total RNAs were isolated and the microRNA and gene expression level was quantified by real time PCR analysis. We found that the expression level of miR-181b in tumor tissues was higher than in normal tissues. Besides, we identified a miR-181b candidate target gene TIMP-3, a tissue metalloproteinase inhibitor, via the TargetScan website. Interestingly, the mRNA levels of miR-181b and TIMP-3 had an opposite behavior in CRC tissues and cell lines. Therefore, we verified the TIMP-3 is a direct target of miR-181b by reporter assay, and miR-181b can reduce the luciferase activity of TIMP-3 through binding to the seed regions on TIMP3 3’-UTR. In addition, the functional role of miR-181b in CRC cell lines were verified by proliferation and apoptosis assays. These data suggest that miR-181b plays a critical role on the regulation of cell proliferation and apoptosis under 5-Fluorouracil (5-FU) treatment. First we found the SW620, with higher miR-181b expression level, was more resistant to 5-FU treatment. And the data suggested that TIMP-3 can induce cell apoptosis and miR-181b can induce proliferation. Most importantly, TIMP-3 could reduce the miR-181b-upregulated proliferation. So we conjectured that the colorectal cancer cell line could be more resistant to 5-FU treatment through TIMP-3 down regulation by miR-181b.Finally, we categorize a rule that the CRC cell lines expressed higher miR-181b level and lower TIMP3 level may be prone to be a drug-resistant cell line, just like the SW620. If the condition is opposite to the former, the cells may be prone to be sensitive to drug treatment. Besides, we think these two factors of miR-181b and TIMP-3 could be good prognostic indicators in the clinical.
論文目次 Chinese abstract I
English abstract III
Acknowledgement V
Contents VII
Figure index X
Introduction
I. The overview of colorectal cancer 1
i. The survival rate of every stages of colorectal cancer 2
ii. The chemotherapeutics of colorectal cancer 2
iii. The mechanism of chemotherapeutic drug (5-FU)-induced cell death 3
II. Introduction of microRNAs 4
i. MicroRNAs and discovery 4
ii. MicroRNA biosynthesis and functions 4
iii. The functional roles of miRNAs in colorectal cancer 5
iv. The miR-181family 6
v. MicroRNA-181b in colorectal cancers 6
III. Tissue inhibitors of metalloproteinases-3 (TIMP-3) 7
i. The overview of Tissue inhibitor of metalloproteinase-3 (TIMP-3) 7
ii. Physical functions of TIMP-3 in colorectal cancer 8
IV. Research aim 9
Materials and Methods
I. Materials 11
II. Primers list 16
III. Methods
A. Cell culture and 5-FU treatment 17
B. Reporter plasmid constructs 17
C. Transient transfection 18
i. Plasmid transfection with PolyJetTM in vitro transfection reagent 18
ii. SiRNA transfection with GenMuteTM siRNA transfection reagent 18
iii. Plasmid transfection with TranIT○R-2020 Transfection Reagent 19
D. RNA extraction 19
E. Preparation protein cell lysates 20
F. Reverse transcription (RT) 20
G. Western blot assay 21
H. Quantitative real-time PCR (qRT-PCR) 21
I. Reporter assay 22
J. Proliferation assay 22
K. Flowcytometry assay 23
Results
I. The expression levels of hsa-miR-181b in tumor tissues were higher than in normal tissues 24
II. The expression levels of miR-181b are correlating with different stages of colorectal cancer cell line 24
i. SW620 cells with higher expression level of miR-181b displays more resistant to 5-FU treatment than SW480 25
ii. The malignant cell lines, SW620, has higher miR-181b level than in SW480 cell lines 25
III. The expression of miR-181b promotes the cell proliferation 26
IV. TIMP-3 3’-UTR contains two possible miR-181b target regions 27
V. The mRNA expression levels of TIMP-3 and miR-181b were opposite in the CRC cell lines 27
i. The RNA expression level of TIMP-3 and miR-181b in CRC samples and cell lines 27
ii. The endogenous TIMP-3 mRNA level was reduced in miR-181b-overexpressed cell lines 28
VI. TIMP-3 RNA levels could be reduced by miR-181b through binding on the seed regions of TIMP-3 3’UTR 28
i. The reporter constructs illustration 28
ii. Reporter assay to confirm the direct binding of miR-181b on TIMP-3 29
VII. To compare the expression fold change between tumor and normal samples of TIMP-3 and miR-181b in human colorectal cancer tissues 29
VIII. TIMP-3 was down-regulated the miR-181b-overexpressed proliferation 30
IX. Apoptosis level of SW620 was induced after TIMP-3 induction and reduced by miR-181b in SW480 30
X. Hypothesis model of this study 31
Discussion
I. The drug resistant phenomenom and colorectal cancer 32
II. The functional roles of miR-181b in human cancers 33
III. The correlation between tissue inhibitor of metalloproteinase-3 (TIMP-3) and 5-FU drug resistance 34
Reference 37
Figures 44
Appendices 55
參考文獻 Akao Y, Noguchi S, Iio A, Kojima K, Takagi T, Naoe T. 2011. Dysregulation of microRNA-34a expression causes drug-resistance to 5-FU in human colon cancer DLD-1 cells. Cancer letters 300(2): 197-204.
Alessandra P. Guglielmi AFS. 2007. Second-Line Therapy for Advanced Colorectal Cancer. Gastrointestinal Cancer Research 1(2): 7.
Arnold CN, Goel A, Blum HE, Boland CR. 2005. Molecular pathogenesis of colorectal cancer: implications for molecular diagnosis. Cancer 104(10): 2035-2047.
Baba Y, Yasuda O, Takemura Y, Ishikawa Y, Ohishi M, Iwanami J, Mogi M, Doe N, Horiuchi M, Maeda N et al. 2009. Timp-3 deficiency impairs cognitive function in mice. Laboratory investigation; a journal of technical methods and pathology 89(12): 1340-1347.
Bond M, Murphy G, Bennett MR, Amour A, Knauper V, Newby AC, Baker AH. 2000. Localization of the death domain of tissue inhibitor of metalloproteinase-3 to the N terminus. Metalloproteinase inhibition is associated with proapoptotic activity. The Journal of biological chemistry 275(52): 41358-41363.
Bond M, Murphy G, Bennett MR, Newby AC, Baker AH. 2002. Tissue inhibitor of metalloproteinase-3 induces a Fas-associated death domain-dependent type II apoptotic pathway. The Journal of biological chemistry 277(16): 13787-13795.
Boni V, Bitarte N, Cristobal I, Zarate R, Rodriguez J, Maiello E, Garcia-Foncillas J, Bandres E. 2010. miR-192/miR-215 influence 5-fluorouracil resistance through cell cycle-mediated mechanisms complementary to its post-transcriptional thymidilate synthase regulation. Molecular cancer therapeutics 9(8): 2265-2275.
Cappell MS. 2005. The pathophysiology, clinical presentation, and diagnosis of colon cancer and adenomatous polyps. The Medical clinics of North America 89(1): 1-42, vii.
Cichocki F, Felices M, McCullar V, Presnell SR, Al-Attar A, Lutz CT, Miller JS. 2011. Cutting edge: microRNA-181 promotes human NK cell development by regulating Notch signaling. Journal of immunology (Baltimore, Md : 1950) 187(12): 6171-6175.
Curtin JC. 2013. Novel drug discovery opportunities for colorectal cancer. Expert Opin Drug Discov.
Davies RJ, Miller R, Coleman N. 2005. Colorectal cancer screening: prospects for molecular stool analysis. Nature reviews Cancer 5(3): 199-209.
Dong Y, Wu WK, Wu CW, Sung JJ, Yu J, Ng SS. 2011. MicroRNA dysregulation in colorectal cancer: a clinical perspective. British journal of cancer 104(6): 893-898.
Doyle VC. 2007. Nutrition and colorectal cancer risk: a literature review. Gastroenterology nursing : the official journal of the Society of Gastroenterology Nurses and Associates 30(3): 178-182; quiz 182-173.
Edwards DR. 2004. TIMP-3 and endocrine therapy of breast cancer: an apoptosis connection emerges. The Journal of pathology 202(4): 391-394.
Fata JE, Leco KJ, Voura EB, Yu HY, Waterhouse P, Murphy G, Moorehead RA, Khokha R. 2001. Accelerated apoptosis in the Timp-3-deficient mammary gland. The Journal of clinical investigation 108(6): 831-841.
Garofalo M, Di Leva G, Romano G, Nuovo G, Suh S-S, Ngankeu A, Taccioli C, Pichiorri F, Alder H, Secchiero P et al. 2009. miR-221&222 Regulate TRAIL Resistance and Enhance Tumorigenicity through PTEN and TIMP3 Downregulation. Cancer Cell 16(6): 498-509.
Grem JL. 2000. 5-Fluorouracil: forty-plus and still ticking. A review of its preclinical and clinical development. Investigational new drugs 18(4): 299-313.
Guo JX, Tao QS, Lou PR, Chen XC, Chen J, Yuan GB. 2012. miR-181b as a potential molecular target for anticancer therapy of gastric neoplasms. Asian Pacific journal of cancer prevention : APJCP 13(5): 2263-2267.
Herszenyi L, Hritz I, Lakatos G, Varga MZ, Tulassay Z. 2012. The behavior of matrix metalloproteinases and their inhibitors in colorectal cancer. International journal of molecular sciences 13(10): 13240-13263.
Iseri OD, Kars MD, Arpaci F, Gunduz U. 2010. Gene expression analysis of drug-resistant MCF-7 cells: implications for relation to extracellular matrix proteins. Cancer chemotherapy and pharmacology 65(3): 447-455.
Janne PA, Mayer RJ. 2000. Chemoprevention of colorectal cancer. The New England journal of medicine 342(26): 1960-1968.
Ji J, Yamashita T, Wang XW. 2011. Wnt/beta-catenin signaling activates microRNA-181 expression in hepatocellular carcinoma. Cell & bioscience 1(1): 4.
Kallio JP, Hopkins-Donaldson S, Baker AH, Kähäri V-M. 2011a. TIMP-3 promotes apoptosis in nonadherent small cell lung carcinoma cells lacking functional death receptor pathway. International Journal of Cancer 128(4): 991-996.
Kallio JP, Hopkins-Donaldson S, Baker AH, Kahari VM. 2011b. TIMP-3 promotes apoptosis in nonadherent small cell lung carcinoma cells lacking functional death receptor pathway. International journal of cancer Journal international du cancer 128(4): 991-996.
Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. 2001. Identification of Novel Genes Coding for Small Expressed RNAs. Science 294(5543): 853-858.
Langner E, Przybylowska K, Trzcinski R, Mik M, Galbfach P, Smolarz B, Romanowicz-Makowska H, Smigileski J, Kulig A, Dziki A. 2010. Loss of hMSH2 gene expression correlates with improved survival in patients with sporadic colorectal cancer. J Genet 89(1): 101-104.
Leco KJ, Waterhouse P, Sanchez OH, Gowing KLM, Poole AR, Wakeham A, Mak TW, Khokha R. 2001. Spontaneous air space enlargement in the lungs of mice lacking tissue inhibitor of metalloproteinases-3 (TIMP-3). Journal of Clinical Investigation 108(6): 817-829.
Lee RC, Feinbaum RL, Ambros V. 1993. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75(5): 843-854.
Lewis BP, Burge CB, Bartel DP. 2005. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120(1): 15-20.
Lin H, Zhang Y, Wang H, Xu D, Meng X, Shao Y, Lin C, Ye Y, Qian H, Wang S. 2012. Tissue inhibitor of metalloproteinases-3 transfer suppresses malignant behaviors of colorectal cancer cells. Cancer gene therapy 19(12): 845-851.
Longley DB, Allen WL, Johnston PG. 2006. Drug resistance, predictive markers and pharmacogenomics in colorectal cancer. Biochimica et biophysica acta 1766(2): 184-196.
Matuo R, Sousa FG, Escargueil AE, Grivicich I, Garcia-Santos D, Chies JAB, Saffi J, Larsen AK, Henriques JAP. 2009. 5-Fluorouracil and its active metabolite FdUMP cause DNA damage in human SW620 colon adenocarcinoma cell line. Journal of Applied Toxicology 29(4): 308-316.
Miller TE, Ghoshal K, Ramaswamy B, Roy S, Datta J, Shapiro CL, Jacob S, Majumder S. 2008. MicroRNA-221/222 confers tamoxifen resistance in breast cancer by targeting p27Kip1. The Journal of biological chemistry 283(44): 29897-29903.
Mylona E, Magkou C, Giannopoulou I, Agrogiannis G, Markaki S, Keramopoulos A, Nakopoulou L. 2006. Expression of tissue inhibitor of matrix metalloproteinases (TIMP)-3 protein in invasive breast carcinoma: relation to tumor phenotype and clinical outcome. Breast cancer research : BCR 8(5): R57.
Nakajima G, Hayashi K, Xi Y, Kudo K, Uchida K, Takasaki K, Yamamoto M, Ju J. 2006. Non-coding MicroRNAs hsa-let-7g and hsa-miR-181b are Associated with Chemoresponse to S-1 in Colon Cancer. Cancer genomics & proteomics 3(5): 317-324.
Salter KH, Acharya CR, Walters KS, Redman R, Anguiano A, Garman KS, Anders CK, Mukherjee S, Dressman HK, Barry WT et al. 2008. An integrated approach to the prediction of chemotherapeutic response in patients with breast cancer. PloS one 3(4): e1908.
Schoffski P. 2004. The modulated oral fluoropyrimidine prodrug S-1, and its use in gastrointestinal cancer and other solid tumors. Anti-cancer drugs 15(2): 85-106.
Siomi H, Siomi MC. 2010. Posttranscriptional Regulation of MicroRNA Biogenesis in Animals. Molecular cell 38(3): 323-332.
Slaby O, Svoboda M, Michalek J, Vyzula R. 2009. MicroRNAs in colorectal cancer: translation of molecular biology into clinical application. Molecular cancer 8: 102.
Smith MR, Kung H, Durum SK, Colburn NH, Sun Y. 1997. TIMP-3 induces cell death by stabilizing TNF-alpha receptors on the surface of human colon carcinoma cells. Cytokine 9(10): 770-780.
Valeri N, Gasparini P, Braconi C, Paone A, Lovat F, Fabbri M, Sumani KM, Alder H, Amadori D, Patel T et al. 2010. MicroRNA-21 induces resistance to 5-fluorouracil by down-regulating human DNA MutS homolog 2 (hMSH2). Proceedings of the National Academy of Sciences of the United States of America 107(49): 21098-21103.
Verstappen J, Von den Hoff JW. 2006. Tissue Inhibitors of Metalloproteinases (TIMPs): Their Biological Functions and Involvement in Oral Disease. Journal of Dental Research 85(12): 1074-1084.
Zhang B, Pan X, Cobb GP, Anderson TA. 2007. microRNAs as oncogenes and tumor suppressors. Developmental biology 302(1): 1-12.
Zheng T, Wang J, Chen X, Liu L. 2010. Role of microRNA in anticancer drug resistance. International journal of cancer Journal international du cancer 126(1): 2-10.
Zhu W, Shan X, Wang T, Shu Y, Liu P. 2010. miR-181b modulates multidrug resistance by targeting BCL2 in human cancer cell lines. International journal of cancer Journal international du cancer 127(11): 2520-2529.

Patrias K, author; Wendling D, editor. (2007), Citing Medicine: The NLM Style Guide for Authors, Editors, and Publishers (2nd ed.). Bethesda (MD): National Library of Medicine (US). Available from: http://www.ncbi.nlm.nih.gov/books/NBK7256/
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