miR-328透過靶向CRK抑制侵襲並伴隨GBM的有利生存

Qi-Sheng Hong; 洪啟盛

請用此 Handle URI 來引用此文件： http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/20384

完整後設資料紀錄

DC 欄位	值	語言
dc.contributor.advisor	俞松良
dc.contributor.author	Qi-Sheng Hong	en
dc.contributor.author	洪啟盛	zh_TW
dc.date.accessioned	2021-06-08T02:46:58Z	-
dc.date.copyright	2017-09-08
dc.date.issued	2016
dc.date.submitted	2017-08-21
dc.identifier.citation	1. Mortality, G.B.D. and C. Causes of Death, Global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death, 1980-2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet, 2016. 388(10053): p. 1459-1544. 2. DALYs, G.B.D. and H. Collaborators, Global, regional, and national disability-adjusted life-years (DALYs) for 315 diseases and injuries and healthy life expectancy (HALE), 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet, 2016. 388(10053): p. 1603-1658. 3. Global Burden of Disease Cancer, C., et al., Global, Regional, and National Cancer Incidence, Mortality, Years of Life Lost, Years Lived With Disability, and Disability-Adjusted Life-years for 32 Cancer Groups, 1990 to 2015: A Systematic Analysis for the Global Burden of Disease Study. JAMA Oncol, 2017. 3(4): p. 524-548. 4. Steeg, P.S., Targeting metastasis. Nat Rev Cancer, 2016. 16(4): p. 201-18. 5. Wen, P.Y. and S. Kesari, Malignant gliomas in adults. N Engl J Med, 2008. 359(5): p. 492-507. 6. Chin, L., et al., Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature, 2008. 455(7216): p. 1061-1068. 7. Paul, C.D., P. Mistriotis, and K. Konstantopoulos, Cancer cell motility: lessons from migration in confined spaces. Nat Rev Cancer, 2017. 17(2): p. 131-140. 8. Sahai, E., Illuminating the metastatic process. Nat Rev Cancer, 2007. 7(10): p. 737-49. 9. Mehlen, P. and A. Puisieux, Metastasis: a question of life or death. Nat Rev Cancer, 2006. 6(6): p. 449-58. 10. Zlotnik, A., New insights on the role of CXCR4 in cancer metastasis. J Pathol, 2008. 215(3): p. 211-3. 11. Stafford, L.J., K.S. Vaidya, and D.R. Welch, Metastasis suppressors genes in cancer. Int J Biochem Cell Biol, 2008. 40(5): p. 874-91. 12. Yang, J.S. and E.C. Lai, Alternative miRNA Biogenesis Pathways and the Interpretation of Core miRNA Pathway Mutants. Molecular Cell, 2011. 43(6): p. 892-903. 13. Tsuchiya, S., Y. Okuno, and G. Tsujimoto, MicroRNA: biogenetic and functional mechanisms and involvements in cell differentiation and cancer. J Pharmacol Sci, 2006. 101(4): p. 267-70. 14. Apicelli, A.J., et al., A non-tumor suppressor role for basal p19ARF in maintaining nucleolar structure and function. Mol Cell Biol, 2008. 28(3): p. 1068-80. 15. Esquela-Kerscher, A. and F.J. Slack, Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer, 2006. 6(4): p. 259-69. 16. Calin, G.A., et al., Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci U S A, 2004. 101(9): p. 2999-3004. 17. Calin, G.A., et al., A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N Engl J Med, 2005. 353(17): p. 1793-801. 18. Grosshans, H., et al., The temporal patterning microRNA let-7 regulates several transcription factors at the larval to adult transition in C. elegans. Dev Cell, 2005. 8(3): p. 321-30. 19. Volinia, S., et al., A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A, 2006. 103(7): p. 2257-61. 20. Bagga, S., et al., Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell, 2005. 122(4): p. 553-63. 21. Calin, G.A. and C.M. Croce, MicroRNA signatures in human cancers. Nat Rev Cancer, 2006. 6(11): p. 857-66. 22. Wei, F., et al., miR-373 Inhibits Glioma Cell U251 Migration and Invasion by Down-Regulating CD44 and TGFBR2. Cell Mol Neurobiol, 2016. 36(8): p. 1389-1397. 23. Peng, G., Y. Liao, and C. Shen, miRNA-429 Inhibits Astrocytoma Proliferation and Invasion by Targeting BMI1. Pathol Oncol Res, 2016. 24. Yang, S.F., et al., Upregulation of miR-328 and inhibition of CREB-DNA-binding activity are critical for resveratrol-mediated suppression of matrix metalloproteinase-2 and subsequent metastatic ability in human osteosarcomas. Oncotarget, 2015. 6(5): p. 2736-53. 25. Qian, Z., et al., MiR-328 targeting PIM-1 inhibits proliferation and migration of pulmonary arterial smooth muscle cells in PDGFBB signaling pathway. Oncotarget, 2016. 7(34): p. 54998-55011. 26. Pan, Y.Z., M.E. Morris, and A.M. Yu, MicroRNA-328 negatively regulates the expression of breast cancer resistance protein (BCRP/ABCG2) in human cancer cells. Mol Pharmacol, 2009. 75(6): p. 1374-9. 27. Ulivi, P., et al., Peripheral blood miR-328 expression as a potential biomarker for the early diagnosis of NSCLC. Int J Mol Sci, 2013. 14(5): p. 10332-42. 28. Feller, S.M., Crk family adaptors-signalling complex formation and biological roles. Oncogene, 2001. 20(44): p. 6348-71. 29. Nishihara, H., et al., Molecular and immunohistochemical analysis of signaling adaptor protein Crk in human cancers. Cancer Lett, 2002. 180(1): p. 55-61. 30. Akagi, T., et al., v-Crk activates the phosphoinositide 3-kinase/AKT pathway in transformation. Proc Natl Acad Sci U S A, 2000. 97(13): p. 7290-5. 31. Iwahara, T., et al., CrkII regulates focal adhesion kinase activation by making a complex with Crk-associated substrate, p130Cas. Proc Natl Acad Sci U S A, 2004. 101(51): p. 17693-8. 32. Linghu, H., et al., Involvement of adaptor protein Crk in malignant feature of human ovarian cancer cell line MCAS. Oncogene, 2006. 25(25): p. 3547-56. 33. Watanabe, T., et al., Adaptor molecule Crk is required for sustained phosphorylation of Grb2-associated binder 1 and hepatocyte growth factor-induced cell motility of human synovial sarcoma cell lines. Mol Cancer Res, 2006. 4(7): p. 499-510. 34. Rodrigues, S.P., et al., CrkI and CrkII function as key signaling integrators for migration and invasion of cancer cells. Mol Cancer Res, 2005. 3(4): p. 183-94. 35. Takino, T., et al., CrkI adapter protein modulates cell migration and invasion in glioblastoma. Cancer Res, 2003. 63(9): p. 2335-7. 36. Wang, L., et al., Signaling adaptor protein Crk is indispensable for malignant feature of glioblastoma cell line KMG4. Biochem Biophys Res Commun, 2007. 362(4): p. 976-81. 37. Chen, C.H., et al., A novel function of YWHAZ/beta-catenin axis in promoting epithelial-mesenchymal transition and lung cancer metastasis. Mol Cancer Res, 2012. 10(10): p. 1319-31. 38. Agarwal, V., et al., Predicting effective microRNA target sites in mammalian mRNAs. Elife, 2015. 4. 39. Nielsen, C.B., et al., Determinants of targeting by endogenous and exogenous microRNAs and siRNAs. RNA, 2007. 13(11): p. 1894-910. 40. Wong, N. and X. Wang, miRDB: an online resource for microRNA target prediction and functional annotations. Nucleic Acids Res, 2015. 43(Database issue): p. D146-52. 41. Hsu, S.D., et al., miRNAMap 2.0: genomic maps of microRNAs in metazoan genomes. Nucleic Acids Res, 2008. 36(Database issue): p. D165-9. 42. Hermanson, M., et al., Platelet-derived growth factor and its receptors in human glioma tissue: expression of messenger RNA and protein suggests the presence of autocrine and paracrine loops. 1992(0008-5472 (Print)). 43. Brennan, C., et al., Glioblastoma subclasses can be defined by activity among signal transduction pathways and associated genomic alterations. PLoS One, 2009. 4(11): p. e7752. 44. Verhaak, R.G., et al., Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell, 2010. 17(1): p. 98-110. 45. Yang, X., et al., MiR-26a contributes to the PDGF-BB-induced phenotypic switch of vascular smooth muscle cells by suppressing Smad1. Oncotarget, 2017. 46. Vehlow, A. and N. Cordes, Invasion as target for therapy of glioblastoma multiforme. Biochim Biophys Acta, 2013. 1836(2): p. 236-44. 47. Zhang, Y., et al., Dissecting dysfunctional crosstalk pathways regulated by miRNAs during glioma progression. Oncotarget, 2016. 7(18): p. 25769-82. 48. Kouri, F.M., C. Ritner, and A.H. Stegh, miRNA-182 and the regulation of the glioblastoma phenotype - toward miRNA-based precision therapeutics. Cell Cycle, 2015. 14(24): p. 3794-800. 49. Matsuda, M., et al., Two species of human CRK cDNA encode proteins with distinct biological activities. Mol Cell Biol, 1992. 12(8): p. 3482-9. 50. Dai, C., et al., PDGF autocrine stimulation dedifferentiates cultured astrocytes and induces oligodendrogliomas and oligoastrocytomas from neural progenitors and astrocytes in vivo. Genes Dev, 2001. 15(15): p. 1913-25. 51. Assanah, M., et al., Glial progenitors in adult white matter are driven to form malignant gliomas by platelet-derived growth factor-expressing retroviruses. J Neurosci, 2006. 26(25): p. 6781-90. 52. Baris, S., et al., Severe Early-Onset Combined Immunodeficiency due to Heterozygous Gain-of-Function Mutations in STAT1. J Clin Immunol, 2016. 36(7): p. 641-8. 53. Khodarev, N.N., et al., Signal transducer and activator of transcription 1 regulates both cytotoxic and prosurvival functions in tumor cells. Cancer Res, 2007. 67(19): p. 9214-20. 54. Khodarev, N.N., et al., STAT1 pathway mediates amplification of metastatic potential and resistance to therapy. PLoS One, 2009. 4(6): p. e5821. 55. Weichselbaum, R.R., et al., An interferon-related gene signature for DNA damage resistance is a predictive marker for chemotherapy and radiation for breast cancer. Proc Natl Acad Sci U S A, 2008. 105(47): p. 18490-5. 56. Pitroda, S.P., et al., STAT1-dependent expression of energy metabolic pathways links tumour growth and radioresistance to the Warburg effect. BMC Med, 2009. 7: p. 68. 57. Khodarev, N.N., et al., STAT1 is overexpressed in tumors selected for radioresistance and confers protection from radiation in transduced sensitive cells. Proc Natl Acad Sci U S A, 2004. 101(6): p. 1714-9. 58. Tsai, M.H., et al., Gene expression profiling of breast, prostate, and glioma cells following single versus fractionated doses of radiation. Cancer Res, 2007. 67(8): p. 3845-52. 59. Duarte, C.W., et al., Expression signature of IFN/STAT1 signaling genes predicts poor survival outcome in glioblastoma multiforme in a subtype-specific manner. PLoS One, 2012. 7(1): p. e29653.
dc.identifier.uri	http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/20384	-
dc.description.abstract	多年來，癌症一直佔據國人十大死因的榜首，對國人健康產生很大的威脅。根據衛生署癌症登記統計，台灣每年大約有六百位原發性惡性腦瘤的新生病例，其中最常見的腦瘤就是多形性膠質母細胞瘤(GBM，Glioblastoma Multiforme)，約佔所有惡性腦瘤的50-60%。GBM同時也是預後最差的原發腦瘤，平均存活率只有15-18個月。一般而言，惡性腦瘤標準的治療是全部或者局部的切除腫瘤之後，進行放射治療以及施予化療藥物。然而治療效果相當有限，因此了解腦瘤發生原因進而研發新的治療方式是勢在必行。微核醣核酸是一種長約20-23 核苷酸的內生性核醣核酸，它可以透過轉錄後機制抑制數以百計下游基因的蛋白質表現。微核醣核酸的異常表現與許多種癌症的某些特徵有關連性。我們研究結果發現微核醣核酸hsa-miR-328與病人存活率有顯著相關。同時發現hsa-miR-328會抑制U251和SNB19 GBM細胞株的侵襲和轉移能力。運用生物資訊工具發現CRK是微核醣核酸hsa-miR-328的潛在標的基因，並利用報導基因分析及西方墨點法證明其間的調控關係，證實微核醣核酸hsa-miR-328可以減少U251和SNB19 CRK的表現量。藉由減少CRK的表現，hsa-miR-328可以抑制神經膠瘤細胞的侵襲和轉移能力。在hsa-miR-328的啟動子(promoter)，我們發現有三個STAT1的可能結合位點，並且利用報導基因分析證明STAT1可以利用其中一個位點抑制hsa-miR-328的表現量。血小板衍生生長因子(Platelet-derived growth factor，PDGF) 可以調控細胞的生長和分化，常常在GBM患者腦瘤部位發現有PDGF的過量表現。我們同時進一步發現PDGF可以藉由刺激STAT1的磷酸化，活化STAT1進而抑制hsa-miR-328的表現量，使得hsa-miR-328的標的基因CRK表現量上升，促進了GBM細胞的侵襲和轉移能力。	zh_TW
dc.description.abstract	Glioblastomas multiforme (GBM) is the most malignant brain tumor because of the high invasive property and poor prognosis of patients. Treatments of patients diagnosed with GBM have been largely ineffective which consist of surgical resection followed by radiation and/or chemotherapy. However, the survival of patients with malignant glioma remains limited. Our present study of the microRNA expression signature in GBM revealed that miR-328 is significantly downregulated in cancer tissues. Kaplan-Meier survival curves showed that low expression of miR-328 predicted a short duration of progression to GBM. Functional studies including trans-well migration and matri-gel invasion were analyzed using two GBM cell lines, U251 and SNB19. Restoration of miR-328 in cancer cells revealed that miR-328 significantly inhibited cancer cell migration and invasion. CRK was directly regulated by the miR-328 in U251 and SNB19 cells. Our data also demonstrated that STAT1 could inhibit miR-328 expression through its promoter binding and PDGF-BB could inhibit miR-328 expression by STAT1 stimulation.	en
dc.description.provenance	Made available in DSpace on 2021-06-08T02:46:58Z (GMT). No. of bitstreams: 1 ntu-105-D97424007-1.pdf: 3080970 bytes, checksum: f1d2445bb64d605e2d17e0f87c5526d3 (MD5) Previous issue date: 2016	en
dc.description.tableofcontents	口試委員會審定書 i 誌謝 ii 中文摘要 iii Abstract v Introduction 1 1.1 Cancer 2 1.2 Brain cancer 2 1.3 Metastasis 3 1.4 microRNA 5 1.5 MicroRNA-328 6 1.6 CRK gene 7 Materials and Methods 8 2.1 Patients and Specimens 9 2.2 miRNA profiling on universal Bead Array platform 10 2.3 Cell Culture 12 2.4 Transfection of cultured glioma cells 12 2.5 Cell proliferation 13 2.6 In vitro migration and matrigel invasion assay 13 2.7 Western blotting 14 2.8 Reporter vectors constructs and Luciferase assay 14 2.9 Quantification of miRNA expression 15 2.10 Statistical analysis 16 Results 18 3.1 Identification and discovery of prognostic microRNA in GBM specimens by miRNA expression signatures 19 3.2 Effects of ectopic miR-328 expression on cell migration, and invasion in U251 and SNB19 cells 20 3.3 Identification of target gene regulated by the miR-328 in GBM 21 3.4 CRK was a direct target of miR-328 in GBM cells 21 3.5 Effects of silencing CRK on cell migration and invasion in GBM cells 22 3.6 PDGF increase CRK expression through suppression miR328 23 3.7 Overexpress miR-328 attenuate CRK expression causing by PDGF 24 Discussion 25 Figures and Tables 29 Table 1. 30 differential expressed miRNAs between tumor and normal tissues. 30 Table 2. Hazard ratios of overall survival for miRNAs 31 Figure 1. Associations between miR-328 expression and brain tumor 32 Figure 2. Associations between miR-328 expression and GBM 33 Figure 3. Endogenous miR-328 expression in GBM cell lines 34 Figure 4. miR-328 inhibits migration activity of U251 and SNB19 cells. 35 Figure 5. miR-328 inhibits invasion activity of U251 and SNB19 cells. 36 Figure 6. Effects of anti-miR-328 transfection on migration activity in U251 and SNB19 cells. 37 Figure 7. Effects of anti-miR-328 transfection on invasion activity in U251 and SNB19 cells. 38 Figure 8. Downregulation of CRK expression by miR-328 in U251 and SNB19 cells. 39 Figure 9. Schematic diagram of the miR-328 target sites of human and other representative mammal CRK 3’UTRs. 40 Figure 10. Schematic diagram of the wild-type and the mutant type of luciferase reporter constructs. 41 Figure 11. Dual-luciferase reporter assays showing miR-328 repress Crk wild-type UTR. 42 Figure 12. Downregulation of exogenous CRK expression by miR-328 transfection in 293T cells. 43 Figure 13. Downregulation of endogenous CRK expression by miR-328 transfection in U251 and SNB19 cells. 44 Figure 14. Efficiency of CRK knowndown by shCRK in U251 cells 45 Figure 15. Effect of silencing CRK expression by sh-CRK transfection on migration activity in U251 and SNB19 cell lines. 46 Figure 16. Effect of silencing CRK expression by si-CRK transfection on invasion activity in U251 and SNB19 cell lines. 47 Figure 17. miR-328 is downregulated in PDGFBB-stimulated U251 and SNB19 cells. 48 Figure 18. Schematic diagram of the miR-328 promoter luciferase reporter constructs. 49 Figure 19. Stat1 suppresses miR-328 through promoter binding. 50 Figure 20. PDGF-BB increase Crk protein expression and overexpress miR-328 could attenuate PDGF-BB-induced Crk expression. 51 Reference 52 Appendices 62
dc.language.iso	en
dc.title	miR-328透過靶向CRK抑制侵襲並伴隨GBM的有利生存	zh_TW
dc.title	miR-328 inhibits invasion and associates with favorable survival of GBM through targeting CRK	en
dc.type	Thesis
dc.date.schoolyear	105-2
dc.description.degree	博士
dc.contributor.oralexamcommittee	陳健尉,楊慕華,蘇剛毅,華國泰
dc.subject.keyword	多形性膠質母細胞瘤,微核醣核酸,信號轉導及轉錄激活蛋白1,血小板衍生生長因子,腦瘤,	zh_TW
dc.subject.keyword	GBM,miR-328,STAT1,PDGF,brain tumor,	en
dc.relation.page	62
dc.identifier.doi	10.6342/NTU201704064
dc.rights.note	未授權
dc.date.accepted	2017-08-21
dc.contributor.author-college	醫學院	zh_TW
dc.contributor.author-dept	醫學檢驗暨生物技術學研究所	zh_TW
顯示於系所單位：	醫學檢驗暨生物技術學系

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