請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18840
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄭永銘(Yung-Ming Jeng) | |
dc.contributor.author | Yu-Hsin Lee | en |
dc.contributor.author | 李宇心 | zh_TW |
dc.date.accessioned | 2021-06-08T01:30:04Z | - |
dc.date.copyright | 2014-10-09 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-07-28 | |
dc.identifier.citation | References
1. Venook AP, Papandreou C, Furuse J, de Guevara LL. The incidence and epidemiology of hepatocellular carcinoma: a global and regional perspective. Oncologist 2010; 15 Suppl 4: 5-13. 2. Center MM, Jemal A. International trends in liver cancer incidence rates. Cancer Epidemiol Biomarkers Prev 2011; 20: 2362-2368. 3. Paterlini-Brechot P, Saigo K, Murakami Y et al. Hepatitis B virus-related insertional mutagenesis occurs frequently in human liver cancers and recurrently targets human telomerase gene. Oncogene 2003; 22: 3911-3916. 4. Block TM, Mehta AS, Fimmel CJ, Jordan R. Molecular viral oncology of hepatocellular carcinoma. Oncogene 2003; 22: 5093-5107. 5. Melen K, Fagerlund R, Nyqvist M et al. Expression of hepatitis C virus core protein inhibits interferon-induced nuclear import of STATs. J Med Virol 2004; 73: 536-547. 6. Siegel AB, Zhu AX. Metabolic syndrome and hepatocellular carcinoma: two growing epidemics with a potential link. Cancer 2009; 115: 5651-5661. 7. Rahman R, Hammoud GM, Almashhrawi AA et al. Primary hepatocellular carcinoma and metabolic syndrome: An update. World J Gastrointest Oncol 2013; 5: 186-194. 8. Sanyal AJ, Yoon SK, Lencioni R. The etiology of hepatocellular carcinoma and consequences for treatment. Oncologist 2010; 15 Suppl 4: 14-22. 9. Thorgeirsson SS, Grisham JW. Molecular pathogenesis of human hepatocellular carcinoma. Nat Genet 2002; 31: 339-346. 10. Galli A, Svegliati-Baroni G, Ceni E et al. Oxidative stress stimulates proliferation and invasiveness of hepatic stellate cells via a MMP2-mediated mechanism. Hepatology 2005; 41: 1074-1084. 11. Feitelson MA, Sun B, Satiroglu Tufan NL et al. Genetic mechanisms of hepatocarcinogenesis. Oncogene 2002; 21: 2593-2604. 12. Yoshida T, Hisamoto T, Akiba J et al. Spreds, inhibitors of the Ras/ERK signal transduction, are dysregulated in human hepatocellular carcinoma and linked to the malignant phenotype of tumors. Oncogene 2006; 25: 6056-6066. 13. Hsu HC, Jeng YM, Mao TL et al. Beta-catenin mutations are associated with a subset of low-stage hepatocellular carcinoma negative for hepatitis B virus and with favorable prognosis. Am J Pathol 2000; 157: 763-770. 14. Qi LN, Bai T, Chen ZS et al. The p53 mutation spectrum in hepatocellular carcinoma from Guangxi, China : role of chronic hepatitis B virus infection and aflatoxin B1 exposure. Liver Int 2014. 15. Guichard C, Amaddeo G, Imbeaud S et al. Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma. Nat Genet 2012; 44: 694-698. 16. Saint-Andre V, Batsche E, Rachez C, Muchardt C. Histone H3 lysine 9 trimethylation and HP1gamma favor inclusion of alternative exons. Nat Struct Mol Biol 2011; 18: 337-344. 17. Kolasinska-Zwierz P, Down T, Latorre I et al. Differential chromatin marking of introns and expressed exons by H3K36me3. Nat Genet 2009; 41: 376-381. 18. Kanai Y, Saito Y, Ushijima S, Hirohashi S. Alterations in gene expression associated with the overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, during human hepatocarcinogenesis. J Cancer Res Clin Oncol 2004; 130: 636-644. 19. Talos F, Abraham A, Vaseva AV et al. p73 is an essential regulator of neural stem cell maintenance in embryonal and adult CNS neurogenesis. Cell Death Differ 2010; 17: 1816-1829. 20. Yea S, Narla G, Zhao X et al. Ras promotes growth by alternative splicing-mediated inactivation of the KLF6 tumor suppressor in hepatocellular carcinoma. Gastroenterology 2008; 134: 1521-1531. 21. Shilo A, Ben Hur V, Denichenko P et al. Splicing factor hnRNP A2 activates the Ras-MAPK-ERK pathway by controlling A-Raf splicing in hepatocellular carcinoma development. RNA 2014; 20: 505-515. 22. Zhou ZJ, Dai Z, Zhou SL et al. Overexpression of HnRNP A1 promotes tumor invasion through regulating CD44v6 and indicates poor prognosis for hepatocellular carcinoma. Int J Cancer 2013; 132: 1080-1089. 23. Amann T, Bataille F, Spruss T et al. Reduced expression of fibroblast growth factor receptor 2IIIb in hepatocellular carcinoma induces a more aggressive growth. Am J Pathol 2010; 176: 1433-1442. 24. Castillo J, Goni S, Latasa MU et al. Amphiregulin induces the alternative splicing of p73 into its oncogenic isoform DeltaEx2p73 in human hepatocellular tumors. Gastroenterology 2009; 137: 1805-1815 e1801-1804. 25. Chen L, Tovar-Corona JM, Urrutia AO. Alternative splicing: a potential source of functional innovation in the eukaryotic genome. Int J Evol Biol 2012; 2012: 596274. 26. Licatalosi DD, Darnell RB. RNA processing and its regulation: global insights into biological networks. Nat Rev Genet 2010; 11: 75-87. 27. Wang ET, Sandberg R, Luo S et al. Alternative isoform regulation in human tissue transcriptomes. Nature 2008; 456: 470-476. 28. Srebrow A, Kornblihtt AR. The connection between splicing and cancer. J Cell Sci 2006; 119: 2635-2641. 29. Jensen CJ, Oldfield BJ, Rubio JP. Splicing, cis genetic variation and disease. Biochem Soc Trans 2009; 37: 1311-1315. 30. Nowak DG, Woolard J, Amin EM et al. Expression of pro- and anti-angiogenic isoforms of VEGF is differentially regulated by splicing and growth factors. J Cell Sci 2008; 121: 3487-3495. 31. Steinman HA, Burstein E, Lengner C et al. An alternative splice form of Mdm2 induces p53-independent cell growth and tumorigenesis. J Biol Chem 2004; 279: 4877-4886. 32. Barbier J, Dutertre M, Bittencourt D et al. Regulation of H-ras splice variant expression by cross talk between the p53 and nonsense-mediated mRNA decay pathways. Mol Cell Biol 2007; 27: 7315-7333. 33. Kim E, Goren A, Ast G. Insights into the connection between cancer and alternative splicing. Trends Genet 2008; 24: 7-10. 34. Berasain C, Castillo J, Prieto J, Avila MA. New molecular targets for hepatocellular carcinoma: the ErbB1 signaling system. Liver Int 2007; 27: 174-185. 35. Plowman SJ, Arends MJ, Brownstein DG et al. The K-Ras 4A isoform promotes apoptosis but does not affect either lifespan or spontaneous tumor incidence in aging mice. Exp Cell Res 2006; 312: 16-26. 36. Grosso AR, Martins S, Carmo-Fonseca M. The emerging role of splicing factors in cancer. EMBO Rep 2008; 9: 1087-1093. 37. Jeong MH, Bae J, Kim WH et al. p19ras interacts with and activates p73 by involving the MDM2 protein. J Biol Chem 2006; 281: 8707-8715. 38. Marcel V, Hainaut P. p53 isoforms - a conspiracy to kidnap p53 tumor suppressor activity? Cell Mol Life Sci 2009; 66: 391-406. 39. Venables JP. Unbalanced alternative splicing and its significance in cancer. Bioessays 2006; 28: 378-386. 40. Berasain C. Impairment of pre-mRNA splicing in liver disease: Mechanisms and consequences. World Journal of Gastroenterology 2010; 16: 3091. 41. David CJ, Manley JL. Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged. Genes Dev 2010; 24: 2343-2364. 42. Lunde BM, Moore C, Varani G. RNA-binding proteins: modular design for efficient function. Nat Rev Mol Cell Biol 2007; 8: 479-490. 43. Du H, Cline MS, Osborne RJ et al. Aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy. Nat Struct Mol Biol 2010; 17: 187-193. 44. Lukong KE, Chang KW, Khandjian EW, Richard S. RNA-binding proteins in human genetic disease. Trends Genet 2008; 24: 416-425. 45. Han H, Irimia M, Ross PJ et al. MBNL proteins repress ES-cell-specific alternative splicing and reprogramming. Nature 2013; 498: 241-245. 46. Begemann G, Paricio N, Artero R et al. muscleblind, a gene required for photoreceptor differentiation in Drosophila, encodes novel nuclear Cys3His-type zinc-finger-containing proteins. Development 1997; 124: 4321-4331. 47. Fardaei M, Rogers MT, Thorpe HM et al. Three proteins, MBNL, MBLL and MBXL, co-localize in vivo with nuclear foci of expanded-repeat transcripts in DM1 and DM2 cells. Hum Mol Genet 2002; 11: 805-814. 48. Charizanis K, Lee KY, Batra R et al. Muscleblind-like 2-mediated alternative splicing in the developing brain and dysregulation in myotonic dystrophy. Neuron 2012; 75: 437-450. 49. Hao M, Akrami K, Wei K et al. Muscleblind-like 2 (Mbnl2) -deficient mice as a model for myotonic dystrophy. Dev Dyn 2008; 237: 403-410. 50. Brook JD, McCurrach ME, Harley HG et al. Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. Cell 1992; 68: 799-808. 51. Liquori CL, Ricker K, Moseley ML et al. Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9. Science 2001; 293: 864-867. 52. Udd B, Krahe R. The myotonic dystrophies: molecular, clinical, and therapeutic challenges. The Lancet Neurology 2012; 11: 891-905. 53. Miller JW, Urbinati CR, Teng-Umnuay P et al. Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy. EMBO J 2000; 19: 4439-4448. 54. Warf MB, Berglund JA. MBNL binds similar RNA structures in the CUG repeats of myotonic dystrophy and its pre-mRNA substrate cardiac troponin T. RNA 2007; 13: 2238-2251. 55. Chaudhuri T, Mukherjea M, Sachdev S et al. Role of the fetal and alpha/beta exons in the function of fast skeletal troponin T isoforms: correlation with altered Ca2+ regulation associated with development. J Mol Biol 2005; 352: 58-71. 56. Kino Y, Washizu C, Oma Y et al. MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1. Nucleic Acids Res 2009; 37: 6477-6490. 57. Ladd AN, Stenberg MG, Swanson MS, Cooper TA. Dynamic balance between activation and repression regulates pre-mRNA alternative splicing during heart development. Dev Dyn 2005; 233: 783-793. 58. Machuca-Tzili LE, Buxton S, Thorpe A et al. Zebrafish deficient for Muscleblind-like 2 exhibit features of myotonic dystrophy. Dis Model Mech 2011; 4: 381-392. 59. Adereth Y, Dammai V, Kose N et al. RNA-dependent integrin alpha3 protein localization regulated by the Muscleblind-like protein MLP1. Nat Cell Biol 2005; 7: 1240-1247. 60. Shapiro IM, Cheng AW, Flytzanis NC et al. An EMT-driven alternative splicing program occurs in human breast cancer and modulates cellular phenotype. PLoS Genet 2011; 7: e1002218. 61. Gabut M, Samavarchi-Tehrani P, Wang X et al. An alternative splicing switch regulates embryonic stem cell pluripotency and reprogramming. Cell 2011; 147: 132-146. 62. Venables JP, Lapasset L, Gadea G et al. MBNL1 and RBFOX2 cooperate to establish a splicing programme involved in pluripotent stem cell differentiation. Nat Commun 2013; 4: 2480. 63. Sakamoto T, Liu Z, Murase N et al. Mitosis and apoptosis in the liver of interleukin-6-deficient mice after partial hepatectomy. Hepatology 1999; 29: 403-411. 64. Amit M, Laevsky I, Miropolsky Y et al. Dynamic suspension culture for scalable expansion of undifferentiated human pluripotent stem cells. Nat Protoc 2011; 6: 572-579. 65. Hong X, Chedid K, Kalkanis SN. Glioblastoma cell line-derived spheres in serumcontaining medium versus serum-free medium: a comparison of cancer stem cell properties. Int J Oncol 2012; 41: 1693-1700. 66. Yoon SM, Gerasimidou D, Kuwahara R et al. Epithelial cell adhesion molecule (EpCAM) marks hepatocytes newly derived from stem/progenitor cells in humans. Hepatology 2011; 53: 964-973. 67. Okabe M, Tsukahara Y, Tanaka M et al. Potential hepatic stem cells reside in EpCAM+ cells of normal and injured mouse liver. Development 2009; 136: 1951-1960. 68. Yamashita T, Ji J, Budhu A et al. EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features. Gastroenterology 2009; 136: 1012-1024. 69. Ren X, Hogaboam C, Carpenter A, Colletti L. Stem cell factor restores hepatocyte proliferation in IL-6 knockout mice following 70% hepatectomy. Journal of Clinical Investigation 2003; 112: 1407-1418. 70. Desmet V, Roskams T, Van Eyken P. Ductular reaction in the liver. Pathol Res Pract 1995; 191: 513-524. 71. Lee KY, Li M, Manchanda M et al. Compound loss of muscleblind-like function in myotonic dystrophy. EMBO Mol Med 2013; 5: 1887-1900. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18840 | - |
dc.description.abstract | 真核細胞mRNA前驅分子經過剪接的過程成為成熟的mRNA,而mRNA前驅分子的選擇性剪接可增加蛋白質的多樣性和基因表達的複雜程度。選擇性剪接不僅影響細胞的分化及個體的發育,目前已知許多遺傳疾病以及癌細胞的產生與轉移都與mRNA的剪接有密切的關係。人類muscleblind-like蛋白質(muscleblind-like protein, MBNL protein)是RNA結合蛋白,可調節RNA的選擇性剪接。近期的研究發現,在胚胎幹细胞中提高muscleblind-like蛋白質的表現量,可誘導分化特性的選擇性剪接事件產生。在我們的實驗中發現,經由免疫組織化學染色法可看到MBNL2會染在肝臟幹細胞所在的區域 (Canal of Herring)以及由肝臟幹細胞所新生成的肝細胞內,MBNL2也會染在具癌前病變特徵的異生性肝臟結節(75%)和肝細胞癌(59.8%)的區域。在統計分析中發現,當腫瘤大小大於5公分時,MBNL2表現量高的比例(25.7%)比小於5公分的比例(49.3%)要少(p=0.004),因此我們認為在肝癌發生的過程中MBNL2的減少會使腫瘤生長的更大。在體外培養實驗中,提高HepJ5肝癌細胞的MBNL2表現量,會減少細胞增殖的速度、聚球(sphere formation)能力、爬行和侵犯的能力,並在小鼠異種移植的試驗中減少腫瘤生長的大小。反之,經由核糖核酸干擾(RNA interference)的方式,剃除HA22T和Huh7肝癌細胞中的MBNL2表現量,在體外培養實驗中會增加細胞爬行和侵犯的能力,但並不足以促進腫瘤的生長。綜合上述的實驗結果,我們證明了MBNL2在肝癌發生的過程中扮演了一個腫瘤抑制蛋白的角色。 | zh_TW |
dc.description.abstract | Pre-mRNA alternative splicing is an essential step in the process of gene expression. It provides cells with the opportunity to create different protein isoforms. Most human genes undergo alternative splicing events, and disruptions of this process have been associated with a variety of diseases, including cancer. The muscleblind protein (MBNL), an RNA binding protein, is a splicing-regulating factor. Recently, it was found that overexpression of MBNL proteins in embryonic stem cells promotes differentiated cell-like alternative splicing patterns. In our study, we performed immunohistochemical staining in liver tissue and hepatocellular carcinoma (HCC), and we found MBNL2 was stained on canal of Herring and hepatocytes newly derived from hepatic progenitor cells. MBNL2 was overexpressed in premalignant dysplastic nodule (75%) and HCC (59.8%). Further statistical analysis showed the percentage of MBNL2-high group in tumor size > 5cm group (25.7%) is less than in tumor size ≦5cm group (49.3%) (p=0.004) , indicating MBNL2 loss might enhance tumor growth in late HCC development stage. Overexpression of MBNL2 in Hep-J5 suppressed proliferation, sphere formation, migration, and invasion in vitro and reduced in vivo tumour growth in NOD/SCID mice. In contrast, depletion of MBNL2 with RNA interference in HCC cell line HA22T and Huh7 caused an increase in migration and invasion in vitro but did not enhance tumor growth. Our results indicate that MBNL2 is a tumor suppressor protein in liver carcinogenesis. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T01:30:04Z (GMT). No. of bitstreams: 1 ntu-103-R01444003-1.pdf: 6261837 bytes, checksum: f35d718793b76173901efb389fe43f16 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | TABLE OF CONTENTS
CHAPTER PAGE 口試委員審定書……………………………………..………………………………. I 謝辭…………………………………………………….……………………………. II 中文摘要 …………………………………………………………………….…..… III ABSTRACT …………………………………………….………………………..… IV I INTRODUCTION ………………………………………………………………… 1 1.1 Hepatocellular carcinoma ………………………………………...………...… 1 1.2 Alternative Splicing and Cancer Development ………………………….....… 2 1.3 The function of MBNL family …………………………………………….…. 5 1.4 Aims of the study ………………………………………………………….…. 7 II MATERIALS AND METHODS………………………………………………… 8 2.1 Immunohistochemistry …………………………………………………….… 8 2.2 Statistical analysis ………………………………………………………….… 9 2.3 Partial hepatectomy model for liver regeneration ………………………….… 9 2.4 Cell culture……………………………………….……………………………10 2.5 Overexpression of MBNL2 …………………………………………………..10 2.6 RNA interference for knockdown experiments …………………………........10 2.7 Western Blotting …………………………………………………………...…11 2.8 MTT assay …………………………………………………………………...12 2.9 Soft agar colony-forming assay ……………………………………………...13 2.10 RNA isolation, RT-PCR and quantitative real-time PCR analysis …………..13 2.11 In vitro sphere formation ………………………………………………….…14 2.12 Xenograft tumor formation in mice …………………………………….……15 2.13 In vitro Boyden Chamber Migration and Invasion assay……………….…….15 III RESULTS………..…………………………………………………………….….17 3.1 Expression of MBNL2 in HCC…………………………………………….......17 3.2 Expression of MBNL2 in hepatocytes newly derived from hepatic progenitor cell …………………………………………………………………………..…17 3.3 Expression of MBNL2 in premalignant dysplastic nodule…………………......18 3.4 Correlation of clinicopathological factors and MBNL2 expression in HCC.......18 3.5 MBNL2 is required for hepatocyte proliferation in vivo………………...…..…19 3.6 MBNL2 overexpression in Hep-J5 inhibited anchorage-independent growth, tumor sphere formation and in vivo tumorigenicity.…………………….…..…20 3.7 MBNL2 overexpression in Hep-J5 suppressed cell migration and invasion…....21 3.8 Knock down of MBNL2 in HA22T and Huh7 did not affect in vitro proliferation, anchorage-independent growth, and tumor formation in mice……………...….21 3.9 Knock down of MBNL2 in HA22T and Huh7 cells enhanced migration and invasion….……………………………………………………………………..22 IV DISCUSSION….…………………………………………………………………..23 V FIGURES AND TABLES…….……………………………………………………28 Figure 1. Immunostaining of MBNL2 in non-tumorous liver parenchyma and HCC..28 Figure 2 . MBNL2 is expressed in newly-formed hepatocytes………………………...29 Figure 3. Immunostaining of MBNL2 in dysplastic nodule……………………………30 Figure 4. MBNL2 expression level and tumor size…………………………………….32 Figure 5. MBNL2 expression in tumor did not affect the survival and recurrence rates of HCC patients…………………………………………………………………33 Figure 6. Ki-67 positive cells in the livers over a time course after PH………………..34 Figure 7. Upregulation of MBNL2 protein in regenerating liver following PH……….35 Figure 8. Analysis of MBNL2 expression in human liver cancer cell lines by Western blotting…………………………….…………………………………………36 Figure 9. Western blotting confirmed MBNL2 overexpression in Hep-J5 cells…….37 Figure 10. In vitro proliferation of Hep-J5 cells as examined by MTT assay………..38 Figure 11. Effects of MBNL2 overexpression on the ability to form colonies on soft agar………………………………………………………………………..40 Figure 12. MBNL2 overexpression in Hep-J5 inhibited tumor sphere formation……42 Figure 13. The tumorigenicity of Hep-J5 implanted subcutaneously to mice………..44 Figure 14. Boyden chamber assay for Hep-J5 cells…………………………………45 Figure 15. Western blotting confirmed knockdown MBNL2 in HA22T and Huh7 cells…………………………………………………………………….….47 Figure 16. Knock down of MBNL2 did not affect in vitro proliferation………………48 Figure 17. Effects of MBNL2 downregulation on the ability to form colonies on soft agar……………..…………………………………………………………..49 Figure 18. The tumorigenicity of HA22T implanted subcutaneously to mice…………50 Figure 19. Boyden chamber assay for HA22T cells……………………………………51 Figure 20. Boyden chamber assay for Huh7 cells……………………………………...52 REFERENCES………………………………………………………………………..53 | |
dc.language.iso | en | |
dc.title | MBNL2及其造成的mRNA的剪接在肝細胞癌發生的角色 | zh_TW |
dc.title | The role of MBNL2 and its regulated mRNA splicing in liver carcinogenesis | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 連晃駿(Huang-Chun Lien),陳彥榮(Yen-Rong Chen),周涵怡(Han-Yi Chou) | |
dc.subject.keyword | 肝癌,muscleblind-like蛋白質,選擇性剪接,肝臟幹細胞,增殖, | zh_TW |
dc.subject.keyword | hepatocellular carcinoma,alternative splicing,muscleblind protein,hepatic progenitor cell,proliferation, | en |
dc.relation.page | 62 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2014-07-28 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 病理學研究所 | zh_TW |
顯示於系所單位: | 病理學科所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-103-1.pdf 目前未授權公開取用 | 6.12 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。