臺灣博碩士論文加值系統

生殖細胞腫瘤（GCT）是由生殖細胞產生的腫瘤。生殖細胞腫瘤可以是良性或惡性的，通常發生在性腺 (卵巢和睾丸)內，性腺外的生殖細胞腫瘤常是胚胎的發育過程中，錯誤細胞遷移過程所產生的結果。
高達80％的惡性生殖細胞腫瘤具有12p 的iso-chromosome，它是生殖細胞腫瘤的特定遺傳標記。我們搜索了the Human Protein Altas，以尋找在染色體12p 可能的oncogene。我們發現FAM60A位於12p11.21，並且只表現在睾丸生殖細胞。在此研究中，我們探討FAM60A在生殖細胞癌的生長以及分化中扮演的角色。我們的研究發現，在生殖細胞腫瘤細胞株Ntera2 D1使用核糖核酸干擾（RNA interference）抑制FAM60A的表現量會抑制細胞增生、聚球（sphere formation）能力和移動的能力。此外，也會造成細胞周期停滯和細胞淍亡增加。另外，我們在Ntera2 D1細胞株穩定表現FAM60A會增加細胞移動以及聚球（sphere formation）能力。FAM60A的表現不影響Ntera2 D1的神經分化。以螢光素酶報告基因檢測系統分析，我們證實FAM60A是轉錄抑制子。透過微陣列基因表現分析結果，我們發現Nodal可能是受FAM60A調控的基因。此外，我們發現GCT比其他癌症細胞株更適用HDAC抑制劑panobinostat和belinostat 治療。

Germ cell tumors (GCTs) are a neoplasm derived from germ cells. Germ cell tumors can be benign or malignant. They usually occur in the gonads (ovary and testis). Germ cell tumors that originate outside the gonads are due to faulty cell migration during development of the embryo.
Up to 80% of malignant GCTs have isochromosome 12p. Iso-chromosome 12p is a specific genetic marker of GCTs. Because the high frequency of gain of genetic material of 12p in testicular GCTs, we searched the Human Protein Atlas for the identification of the candidate oncogene in chromosome 12p. We identified a gene Family with Sequence Similarity 60 Member A (FAM60A) is a testicular germ cell-specific gene located at 12p11.21. In this study, we examined the roles of FAM60A in GCT growth and differentiation. We found that knockdown of FAM60A in GCT cell line Ntera2 D1 by shRNA suppressed cell proliferation, cell migration and sphere formation in vitro. Besides, it also caused cell cycle arrest and apoptosis. Ntera2 D1 cells stably transfected with FAM60A-expressing plasmid (Ntera2 D1-FAM60A) had enhanced cell migration and sphere formation ability as compared to vector control. The expression of FAM60A did not affect ability of neuronal differentiation in Ntera2 D1 cells. By using a luciferase reporter system, we found that FAM60A is a transcription repressor. Through microarray analysis, we identified that Nodal is a candidate gene regulated by FAM60A. We also found that GCTs are more susceptible to histone deacetylase (HDAC) inhibitors panobinostat and belinostat.

口試委員審定書 I
中文摘要 II
Abstract IV
Index VI
I.Introduction 1
1.1.Germ cell tumor 1
1.2.Morphologic classification of GCT 1
1.3.Chromosomal abnormality in testicular cancer 3
1.4.Family with Sequence Similarity 60 Member A 4
1.5.Aim of the study 5
II.Materials and Methods 6
2.1. Materials 6
2.2. Immunohistochemistry 6
2.3. Cell culture 6
2.4. RNA interference for knockdown experiments 7
2.5. Plasmids and lentiviral infection 8
2.6. RNA isolation 9
2.7. RT-PCR 9
2.8. Real-time PCR 10
2.9. Western Blotting 10
2.10. In vitro sphere formation 11
2.11. Wound-healing Assay 12
2.12. In vitro Boyden chamber migration assay 12
2.13. Cell cycle analysis 13
2.14. Apoptosis assay 13
2.15. Reporter assay 13
2.16. MTT assay 14
2.17. Microarray analysis 14
III. Results
3.1. Specific expression of FAM60A in germ cell tumors 20
3.2. Knockdown of FAM60A in Ntera2 D1 inhibited anchorage-dependent growth, tumor sphere formation, and migration. 20
3.3. Knockdown of FAM60A in Ntera2 D1 cells induced cell cycle arrest and cell apoptosis 21
3.4. Overexpression of FAM60A in Ntera2 D1 cells promoted anchorage-dependent growth, tumor sphere formation and migration 22
3.5. Differentiation of Ntera2 D1 cells and expression analysis of stemness and various neural markers 22
3.6. FAM60A was increased at early time points during differentiation 24
3.7. The knockdown or overexpression of FAM60A did not affect stemness and various neuronal genes expression pattern of Ntera2 D1 cells 24
3.8. Identification of differential gene expression in FAM60A-depleted cells 25
3.9. FAM60A is a transcription repressor and regulates the expression of gene involved in TGF-β signaling pathway 25
3.10. Panobinostat and belinostat cytotoxicity in germ cell lines 26
IV. Discussion 27
4.1. FAM60A is a protein specific for GCT. 27
4.2. FAM60A plays a critical role in growth of GCT 27
4.3. FAM60A gene silencing resulted in cell cycle arrest of Ntera2 cells in G2/M phase. 27
4.4. FAM60A overexpression did not affect stemness and cell differentiation 28
4.5. The novel target genes of FAM60A 29
4.6. The function of Nodal 30
4.7. Panobinostat and belinostat cytotoxicity in germ cell lines 31
V. Figures 32
Figure 1. FAM60A expression in normal testes specimen examined by immunohistochemical stain.. 32
Figure 2. Immunohistochemical stain of FAM60A expression in seminoma specimens. 33
Figure 3. Immunohistochemical stain of FAM60A expression in germ cell tumor specimens. 34
Figure 4. Immunohistochemical stain of FAM60A expression in non-germ cell tumor specimens. 35
Figure 5. Knockdown of FAM60A reduced anchorage-dependent proliferation of Ntera2 D1 cells. 39
Figure 6. Knockdown of FAM60A inhibited tumor sphere formation ability of Ntera2 D1 cells in serum-containing medium. 40
Figure 7. Knockdown of FAM60A inhibited tumor sphere formation ability of Ntera2 D1 cells in serum-free medium. 41
Figure 8. FAM60A knockdown in Ntera2 D1 cells inhibited cell migration in wound-healing assay. 42
Figure 9. FAM60A knockdown in Ntera2 D1 cells inhibited cell migration in Boyden chamber assay. 43
Figure 10. Knockdown of FAM60A in Ntera2 D1 cells induced cell cycle arrest at G2/M phase. 45
Figure 11. Knockdown of FAM60A in Ntera2 D1 cells induced cell cycle arrest at G2/M phase and reduction of cyclin B1 expression. 47
Figure 12. Silencing of FAM60A expression in Ntera2 D1 cells induced apoptosis. 48
Figure 13. Overexpression of FAM60A didn’t not affect cell proliferation. 50
Figure 14. Overexpression of FAM60A promoted tumor sphere formation ability of Ntera2 D1 cells in serum-containing medium. 52
Figure 15. Overexpression of FAM60A promoted tumor sphere formation ability of Ntera2 D1 cells in serum-free medium. 54
Figure 16. FAM60A overexpression in Ntera2 D1 cells promoted cell migration in wound-healing assay. 55
Figure 17. Formation of neurospheres by differentiation of human Ntera2 D1 cells in both adherent and suspension culture condition. 56
Figure 18. Expression pattern of various neuronal and stemness genes in Ntera2 D1 cells differentiation induced by all-trans retinoic acid in adherent culture condition. 58
Figure 19. Expression pattern of various neuronal and stemness genes in Ntera2 D1 cells differentiation induced by all-trans retinoic acid in suspension culture condition. 61
Figure 20. The expression of FAM60A is increased at early time points during Ntera2 D1 cells differentiation induced by all-trans retinoic acid in adherent culture condition. 63
Figure 21. FAM60A expression was increased during Ntera2 D1 cells differentiation induced by all-trans retinoic acid in suspension culture condition. 64
Figure 22. Knockdown of FAM60A expression after all-trans retinoic acid treatment did not affect the expression of stemness gene. 65
Figure 23. Formation of neurospheres by differentiation of FAM60A knocked down cells in adherent culture condition. 67
Figure 24. Overexpression of FAM60A after all-trans retinoic acid treatment did not affect the expression of stemness gene. 68
Figure 25. Formation of neurospheres by differentiation of FAM60A-overepxressing cells in adherent culture condition. 69
Figure 26. Formation of neurospheres by differentiation of FAM60A-overexpressing cells in suspension culture condition. 70
Figure 27. The overexpression of FAM60A did not affect the expression of various neuronal and stemness genes after all-trans retinoic acid treatment in suspension culture condition. 73
Figure 28. Validation of the genes regulated by FAM60A knockdown identified by microarray analysis. 76
Figure 29. FAM60A is a transcription repressor. 77
Figure 30. FAM60A regulates the expression of gene involved in TGF-β signaling pathway. 79
Figure 31. Panobinostat and belinostat decreased germ cells proliferation. 81
Table 1. The clones of shRNA in lentiviral vectors used for knockdown of the endogenous FAM60A 16
Table 2. The primers used for PCR cloning 16
Table 3. The primers used for PCR reaction 17
Table 4. The primers used for PCR reaction 18
Table 5. The primers used for real time-PCR reaction to validating microarray result 19
Table 6. Summary of the expression of FAM60A in the individual types of GCTs and non-germ cell tumors. 37
Table 7. The gene regulated in FAM60A-depleted cells detected by microarray analysis 74
VI. References 83

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