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研究生：	楊逸善 Yang, Yi-Shan
論文名稱：	Protein Y抑制NDPK-A增進神經母細胞瘤侵襲轉移能力 Protein Y counteracts the enhancing ability of NDPK-A in neuroblastoma cell invasiveness
指導教授：	張玲 Chang, Christina Ling
學位類別：	碩士 Master
系所名稱：	醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2016
畢業學年度：	104
語文別：	英文
論文頁數：	60
中文關鍵詞：	核苷二磷酸激酶A 、蛋白質Y 、神經母細胞瘤
外文關鍵詞：	NDPK-A, Protein Y, neuroblastoma
相關次數：	點閱：101 下載：0
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人類的核苷二磷酸激酶A (Nucleotide diphosphate kinase A, NDPK-A) 是第一個被發現與癌症轉移有相關的蛋白質，其由 nm23-H1 基因所編碼。核苷二磷酸激酶 A在小鼠的黑色素瘤以及人類的乳腺癌中扮演著抑制腫瘤轉移的角色。相反的，我們實驗室發現核苷二磷酸激酶 A (Nucleotide diphosphate kinase A, NDPK-A)與其基因變異核苷二磷酸激酶AS120G (NDPK-AS120G)是促進兒童神經母細胞瘤的轉移。到目前為止，核苷二磷酸激酶 A 對腫瘤轉移的機制尚不清楚。我們實驗室最進發現蛋白質Y (Protein Y)與核苷二磷酸激酶A交互作用，但無法與核苷二磷酸激酶AS120G交互作用。在本研究發現在三株神經母細胞瘤之人類細胞株中，意料發現蛋白質Y的mRNA突變產生stop codon因而表現不正常的蛋白質Y。進一步過量表現蛋白質Y在神經母細胞瘤NB69 中，同時表達核苷二磷酸激酶 A或是變異核苷二磷酸激酶AS120G 進行實驗。
Trans-well試驗中，核苷二磷酸激酶 A顯著提高細胞在二度空間的遷移能力，但蛋白質 Y大幅抑制此能力，而兩者則互相增減此能力。但是蛋白質 Y並不抑制變異核苷二磷酸激酶AS120G 遷移能力。在clonogenic試驗中，核苷二磷酸激酶A顯著提高兒童神經母細胞瘤的colony formation能力，但是蛋白質Y則降低此能力，而兩者則彼此增減此能力。當knockdown 蛋白質Y 發現部分回復侵襲轉移能力，相似現象發現在當蛋白質Y的N端被刪除後，僅缺乏N端的蛋白質Y則喪失抑制核苷二磷酸激酶A之能力。並且在斑馬魚 transplantation model 發現到，蛋白質Y不能抑制核苷二磷酸激酶AS120G神經母細胞瘤的侵襲能力。本研究在斑馬魚的發育過程中，利用whole mount in-situ hybridization 發現到nme2b.2 與蛋白質Y 似乎並不參與在斑馬魚神經分化成交感神經的過程。總結以上結果，蛋白質Y具有抑制核苷二磷酸激酶 A 所提高的侵襲轉移能力，但對於核苷二磷酸激酶AS120G 並不能有效的抑制。並且推斷三株神經母細胞瘤可能因蛋白質Y無法正常表現，因而無法抑制核苷二磷酸激酶A對腫瘤轉移的促進能力。

Nucleotide diphosphate kinase A (NDPK-A) is encoded by the nm23-H1/nme1 gene, and acts as a metastasis promoter in neuroblastoma. Overexpression or S120G mutation of NDPK-A found in advanced neuroblastoma increases both invasive and metastatic potential of neuroblastoma cells. However, the molecular mechanism by which NDPK-A promotes neuroblastoma metastasis remains poorly understood. Our lab has found a novel protein, termed Protein Y, that interacted with NDPK-A, but not NDPK-AS120G. In this study, non-sense mutations of the Protein Y mRNA were found in human neuroblastoma NB69, IMR32 and SH-SY5Y cell lines. In stable transducents established from NB69 cells, ectopic expression of NDPK-A or NDPK-AS120G increased cell migration by more than two folds, whereas ectopic expression of Protein Y decreased cell migration by 47% in the trans-well assay. Co-expression of Protein Y completely abolished the migration-enhancing ability of NDPK-A. In contrast, Protein Y displayed little effect on the migration-enhancing ability of NDPK-AS120G. In the clonogenic assay, Protein Y reduced the colony number by 17%, whereas NDPK-A or NDPK-AS120G increased the colony number by 20-25%. Co-expressed Protein Y almost completely abolished the colony-enhancing ability of NDPK-A, but not NDPK-AS120G. While knocking down Protein Y expression partially restored the invasiveness-enhancing ability of NDPK-A, deletion of the N-terminus abolished the ability of Protein Y to counteract the invasiveness-enhancing ability of NDPK-A and, to a lesser degree, NDPK-AS120G. In a zebrafish transplantation model, Protein Y did not seem to affect the extravasation ability of NB69 cells, nor suppress the extravasation-enhancing ability of NDPK-AS120G. By whole mount in-situ hybridization, zebrafish NDPK-A and Protein Y orthologs, namely nme2b.2 and Protein Y, did not appear to participate in the differentiation of neural crest into sympathetic neurons, relevant to neuroblastoma. In conclusion, Protein Y counteracted the ability of NDPK-A and, to a lesser degree, NDPK-AS120G in promoting the invasiveness of NB69 cells. However, such a counteracting ability of Protein Y is likely abrogated by mutation-caused truncations, as detected in its mRNA in human neuroblastoma cell lines examined.

Introduction	1
1	Tumor metastasis	1
2	Neuroblastoma	1-2
3	NDPK-A	2
4	The Puf family	2-3
5	Protein Y	3
6	Zebrafish and applications	3-4
7	Zebrafish orthologs of human NDPK-A and Protein Y	4
Objectives & Specific Aims	5
Materials and Methods	6
1	Cell lines and culture	6
2	Generation of stable transducents that express NDPK-A and/or Protein Y variants	6-7
3	Knockdown of Protein Y expression in stNB-W cells 	7
4	Immunoblotting	7-8
5	Reverse transcription (RT)-PCR	8-9
6	Genomic DNA extraction and amplification		9
7	The MTT assay	9
8	Cell proliferation rate determination	10
9	The clonogenic assay	10
10	Wound healing and trans-well assays	10-11
11	Zebrafish maintenance	11
12	Cloning and generating zebrafish Protein Y and nme2b2 probes	11-12
13	Whole mount in-situ hybridization (WISH)	12
14	Zebrafish transplantation	12-13
15	Immunocytochemistry	13
Results
1	N-terminus deletion of Protein Y does not affect its stability in human neuroblastoma NB69 cells		14
2	Altered subcellular location of Protein Y in certain human neuroblastoma cell lines	14-15
3	The effects of Protein Y and/or NDPK-A variants on the migration of NB69 derivatives 	15-16
4	The effects of Protein Y and/or NDPK-A variants on the survival of NB69 derivatives	16-17
5	The effects of Protein Y and /or NDPK-A variants on human neuroblastoma cell proliferation 	18
6	Mutations occur in Protein Y mRNA in human neuroblastoma cell lines	18-19
7	Knockdown of Protein Y expression decrease the inhibition of cell colony formation and migration ability	19-20
8	Effects of Protein Y on the invasiveness of human neuroblastoma NB69 cells in a zebrafish transplantation model		20
9	Spatial-temporal expression of zebrafish orthologs of human NDPK-A and Protein Y during early development		21
Discussion	22-24
References	25-27
Table 1. The doubling time of NB69 derivatives expressing NDPK-A and/or Protein Y variants	28
Figures
1	Figure 1		29-30
2	Figure 2		31
3	Figure 3		32
4	Figure 4		33
5	Figure 5		34-35
6	Figure 6		36
7	Figure 7		37
8	Figure 8		38-39
9	Figure 9		40
10	Figure 10	41-42
11	Figure 11		43-44
12	Figure 12	45-47
Appendixes
1	Appendix 1. List of antibodies used in this study	48
2	Appendix 2. List of buffers for whole mount in-situ hybridization	48
3	Appendix 3. PCR primers for amplifying exons 2, 3, 4, 5 and 18 in the human Protein Y gene	49
4	Appendix 4. Sequence alignment of RT-PCR products of Protein Y mRNA in five human cancer cell lines	50-51
5	Appendix 5. Alignment the total RT-PCR translation amino acid sequences of the five human cancer cell Protein Y	52-54
6	Appendix 6. Sequence alignment of Protein Y mRNA after cloning of RT-PCR products in four human cell lines	55-58
7	Appendix 7. IMR32 Protein Y mRNA 5’ region direct sequencing PCR product and two different TA clone construct sequencing found mutation	59
8	Appendix 8. Sequence alignment of exons 2, 3, 4 and 18 in Protein Y gene in four human cell lines	60

                                    

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校內：2021-02-15公開
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