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系統識別號 U0026-2305201312562600
論文名稱(中文) 探討與腫瘤轉移相關的NDP kinase A的交互作用蛋白及其特性
論文名稱(英文) Identification and Characterization of An Interacting Protein of Metastasis-Associated NDP Kinase A
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 102
學期 2
出版年 103
研究生(中文) 許人凱
研究生(英文) Jen-Kai Hsu
學號 T16001061
學位類別 碩士
語文別 英文
論文頁數 33頁
口試委員 口試委員-蔣輯武
口試委員-王憲威
指導教授-張玲
中文關鍵字 轉移  NDPK-A  Protein Y  神經母細胞瘤 
英文關鍵字 Metastasis  NDPK-A  Protein Y  neuroblastoma 
學科別分類
中文摘要 腫瘤轉移一直是癌症病患死亡的主因。在人類中發現核苷二磷酸激酶 A (Nucleotide diphosphate kinase A, NDPK-A) 是第一個發現與癌症轉移有相關的,並且是由 nm23-H1 基因所編碼。在小鼠的黑色素瘤以及人類的乳腺癌之中知道核苷二磷酸激酶 A 扮演著抑制腫瘤轉移的角色。相反的,在我們實驗室發現核苷二磷酸激酶 A在兒童神經母細胞瘤中是促進腫瘤的轉移。除了高量表現外,我們實驗室發現在21%轉移的兒童神經母細胞瘤病患中核苷二磷酸激酶 A有 S120G 突變。核苷二磷酸激酶 A 的高量表現與突變使得神經的發育受到阻礙也增加兒童神經母細胞瘤的侵犯性以及在小鼠上腫瘤轉移的能力。到目前為止核苷二磷酸激酶 A 對轉移的機制還尚不清楚。在本研究中,蛋白質 Y ( Protein Y) 被發現到與核苷二磷酸激酶 A有交互作用。蛋白質 Y 在 HeLa 以及 HEK293T 細胞株中的位置在核仁。然而,在人類兒童神經母細胞瘤細胞株 (NB69) 中蛋白質 Y卻不在於核仁。在NB69細胞中外源性蛋白質 Y 會回到核仁的位置,暗示著在NB69細胞株之中蛋白質Y可能有突變或是缺失。我建立異種移植斑馬魚模式平台發現,野生型與S120G突變的核苷二磷酸激酶 A會促進NB69的侵入性,此結果與已往老鼠的實驗相似。然而,抑制了核苷二磷酸激酶 A ,即H118F突變會降低約40%的NB69 侵入性。此外,在班馬魚模式中野生型外源性蛋白質Y不會影響由NDPK-A 所引起的細胞侵入能力。這個發現說明蛋白質Y與核苷二磷酸激酶 A 有交互作用,但可能不參與在由核苷二磷酸激酶 A 調控的腫瘤轉移。
英文摘要 Metastasis remains as a major cause of death in cancer patients. Nucleotide diphosphate kinase A (abbreviated as NDP kinase A or NDPK-A), encoded by the nm23-H1 gene, is the first metastasis-associated protein identified in humans. NDPK-A acts as a metastasis suppressor in murine melanoma and human breast carcinoma. In contrast, our lab reported that NDPK-A functions as a metastasis promoter in neuroblastoma. In addition to overexpression, our lab has detected the S120G mutation of NDPK-A in 21% of patients with advanced stages of neuroblastoma. These NDPK-A alterations not only abrogate neuronal differentiation and increase invasiveness of neuroblastoma cells, but also promote tumor metastasis in mice. To date, the molecular mechanism of NDPK-A in metastasis is unclear. Previusly, our lab has identifies Protein Y as an NDPK-A interacting protein based on the yeast two-hybrid system. In this study, Protein Y was localized to the nucleolus of human cervical cancer HeLa and human embryonic kidney HEK293T cells. However, such nucleolar localization was not present in human neuroblastoma NB69 cells. Ectopic Protein Y was able to restore its nucleolar localization in NB69 cells. However, deletion of the N-terminus and not C-terminus of Protein Y abrogated its nucelolar localization in NB69 cells. Etoposide or serum deprivation did not significantly affect the subcellular localization of Protein Y and its deletion mutants in NB69 derivaties that expressed ectopic NDPK-A. The interaction of Protein Y and NDPK-A was confirmed by immuno-precipitation experiment. I further established a xenotransplant zebrafish model and found that expression of ectopic wild-type and S120G mutation of NDPK-A promoted the invasiveness of NB69 cells in zebrafish, similar to that reported in xenograft mice. When the phosphotransferase activity of NDPK-A was inactivated by the H118F mutation, NB69 cells reduced their invasiveness by ~40% in xenotransplated fish. However, ectopic expression of Protein Y did not affect NDPK-A mediated invasiveness of NB69 cells in zebrafish. These findings indicate that Protein Y interacted with NDPK-A, but might not participate in NDPK-A mediated tumor metastasis.
論文目次 Table of Contents
1. Introduction
1.1 Neuroblastoma and Genetic Alterations .................................1
1.2 NDPK Family ………………….………………...……….…..2
1.3 NDPK-A and Metastasis …………………………………….3
1.4 PUF Protein Family ………….………………………………3
1.5 Human PUF Proteins ………………………………………..4
1.6 Zebrafish Transplantation Model ……………………...…...5
1.7 Study Objectives ……………………………………………..6
2. Materials and Methods
2.1 Cell culture …………………………………………………...6
2.2 Construction of Protein Y expression plasmids in a lentiviral system ………………………………………………………...6
2.3 Generation of stable transducents expressing wild-type Protein Y and its varients ……………………………………7
2.4 Immunoprecipitation ………………………….……………..8
2.5 Western blot analysis ……… ………………………………..9
2.6 Immunocytochemistry ……………………………………….9
2.7 Zebrafish maintainence……………………………………...10
2.8 Xenotransplantation of zebrafish…………………………...10
3. Experiment Results
3.1 Altered subcellular location of Protein Y in human neuroblastoma cell lines ………………………………….….11
3.2 Subcellular localization of Protein Y is not altered by etoposide treatment and serum deprivation in NB69-derived transducents …………………………………………………13
3.3 Protein Y interacts with NDPK-A…………………………...13
3.4 Ectopic NDPK-A or its variants increases the invasiveness of human neuroblastoma NB69 cells in zebrafish ……………14
3.5 Protein Y does not seem to invovle in NDPK-A mediated NB69 cell invasion in zebrafish ……………………………..15
4. Discussion…………………………………………………………..16
5. References……………………………………………………….....18
6. Figures
6.1 Figure 1 ………...……………………………………………..23
6.2 Figure 2 ………………………………………...…………….24
6.3 Figure 3 …………………………………………...………….26
6.4 Figure 4 ……………………………………………..………..27
6.5 Figure 5 ………………………………………………………28
6.6 Figure 6 ………………………………………………………29
6.7 Figure 7 ………………………………………………………30
6.8 Figure 8 ………………………………………………………32
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