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研究生：	林建緯 Lin, Chien-Wei
論文名稱：	以缺氧調控二磷酸腺核糖基化因子調節蛋白之表達於細胞增生與遷移之研究 Regulate the expression of ARF regulators by hypoxia to modulate cell proliferation and migration
指導教授：	李純純 Li, Chun-Chun
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 生命科學系 Department of Life Sciences
論文出版年：	2019
畢業學年度：	107
語文別：	中文
論文頁數：	67
中文關鍵詞：	鳥糞嘌呤核苷酸置換蛋白、缺氧、細胞爬行、細胞增生
外文關鍵詞：	BIGs, hypoxia, cell migration, cell proliferation
相關次數：	點閱：80 下載：1
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在缺氧環境中，缺氧誘發因子(Hypoxia-inducible factors)容易累積並促使缺氧相關基因進行轉錄。鳥糞嘌呤核苷酸交換因子BIG1和BIG2 (Brefeldin A inhibited guanine nucleotide exchange factor)作為二磷酸腺苷核糖基化因子的調節蛋白能促使將GDP置換成GTP藉以活化第一型二磷酸腺苷核糖基化因子 (ADP-ribosylation factor 1)。BIG1與BIG2參與囊泡運輸、高基氏體結構的穩定，也調控肌動蛋白的重組和細胞的活動。缺氧情形誘使胞外囊泡的釋出，藉以釋放血管新生相關蛋白來促進上皮-間質型態轉換與血管新生。過去研究指出，BIG1的缺失會促進神經膠質母細胞瘤的爬行，而在人類乳突甲狀腺癌中，miR-215藉由下調BIG1藉此抑制癌細胞的增生。然而，BIG1與BIG2是否會受到缺氧的調控目前尚未釐清。在本篇論文中，我們研究在缺氧誘發化學藥物DMOG及CoCl2的作用下是否會影響細胞內 BIG1與BIG2的表現量。我們以shRNA的實驗方法獲得能穩定低表現BIG1與BIG2的細胞株來觀察BIG1及BIG2在細胞的型態、移動能力、增生能力以及細胞週期的進程中所扮演的角色。我們也以CRIPSR/Cas9技術敲除BIG1及BIG2的表現，並評估是否會影響細胞增生。總結而言，我們的研究數據提供缺氧環境下對於BIG1及BIG2的表現影響，以及對於細胞遷移與增殖等行為能力所扮演之角色。

Hypoxia-inducible factors (HIFs) which accumulated in hypoxia promote transcription of the hypoxia-related gene. Brefeldin A (BFA)-inhibited guanine nucleotide-exchange factors (BIGs) 1 and 2 function as ARF regulators to accelerate the replacement of GDP-bound with GTP to activate ADP-ribosylation factor 1 (ARF1). BIG1 and BIG2 participate in vesicle trafficking, Golgi structure stabilities, actin remodeling and cell motility. Hypoxia induces the extracellular vesicle secretion to release the angiogenesis related molecule to promote angiogenesis and epithelial-mesenchymal transition. It has been reported that BIG1 knockdown promotes glioblastoma U251 cell migration and down-regulation of BIG1 by miR-215 inhibited papillary thyroid cancer cell proliferation. It remains unknown whether BIG1 and BIG2 could be regulated by hypoxia. In this study, we assess that expression of BIG1 and BIG2 when treatment with the hypoxic mimetic agents, DMOG and CoCl2. We also generated the stably BIG1- or BIG2-knockdown cells by short hairpin RNA (shRNA) to investigate the functional roles of BIG1 or BIG2 in cell morphology, cell motility, cell proliferation and cell cycle progression. Effects of depletion of BIG1 or BIG2 by using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 on cell proliferation was also determined. Taken together, our data could provide information of expression of BIG1 and BIG2 under hypoxia and the roles of BIG1 and BIG2 on cell migration and cell proliferation.

摘要	II
ABSTRACT	III
致謝	IV
TABLE OF CONTENTS	V
TABLE DIRECTORY	VIII
FIGURE DIRECTORY	IX
ABBREVIATION	X
INTRODUCTION	1
1.1	Cancer	1
1.2	Small GTPase	1
1.3	ADP-ribosylation factor (ARF)	2
1.4	Brefeldin A -inhibited guanine nucleotide exchange factor BIG1 and BIG2	3
1.5	Cell proliferation and cell cycle	3
1.6	Hypoxia	5
1.7	Research motivation	6
MATERIALS AND METHODS	8
2.1	Cell culture	8
2.2	Small interfering RNA (siRNA) transfection	8
2.3	RNA isolation	8
2.4	Reverse transcription-PCR (RT-PCR)	8
2.5	Quantitative real-time PCR (qRT-PCR)	9
2.6	Cell lysate preparation	9
2.7	Western blotting	10
2.8	Lentivirus production	10
2.9	Lentivirus transduction	11
2.10	CRISPR lentivirus production	11
2.11	CRISPR lentivirus induction	11
2.12	Cell viability assay	12
2.13	Colony formation assay	12
2.14	Cell morphological analysis	12
2.15	CoCl2 or DMOG induced hypoxia	13
2.16	Exposure of cells to hypoxia	13
2.17	DNA methylation inhibitor 5’-azacytidine and HDAC inhibitor sodium butyrate under hypoxia	13
2.18	Transwell migration assay	14
2.19	Cell cycle in flow cytometry	14
2.20	Analysis of cell migration by wound-healing assay	15
2.21	Animal holding	15
2.22	Xenograft	15
2.23	UALCAN Analysis Based on TCGA Data Set	15
RESULTS	17
3.1 Expression of BIG1 and BIG2 under hypoxia	17
3.2 Effects of DNA methylation inhibitor 5’-azacytidine and HDAC inhibitor sodium butyrate treatment on the expression of BIG1 and BIG2 in U251 cells under normoxia or hypoxia.	17
3.3 The BIG1 and BIG2 expression in TCGA database of different cancers	18
3.4 Depletion of BIG1 or BIG2 in U251 cells by lentiviral shRNA infection	18
3.5 Effects of BIG1 or BIG2 depletion on cell morphology	19
3.6 Effects of BIG1 or BIG2 depletion on cell migration	19
3.7 Effects of BIG1 or BIG2 depletion on cell proliferation	20
3.8 Effects of BIG1 or BIG2 depletion on cell cycle	20
3.9 Effects of BIG1 or BIG2 depletion on gene expression	21
DISCUSSION	22
4.1 BIG1 and BIG2 expression is upregulated in most of human tumor types	22
4.2 The roles of BIG1 and BIG2 in hypoxia	23
4.3 Roles of hypoxia on epigenetic regulation	23
4.4 Roles of ARFGEFs in cell proliferation	24
4.5 Roles of ARFGEFs in cell migration	25
4.6 Activity of ADP-ribosylation factors under hypoxia	26
4.7 Conclusion	26
REFERENCE	27
TABLES	32
FIGURES	34
APPENDICES	61


 
TABLE DIRECTORY

Table 1. shRNA sequence	32
Table 2. siRNA sequence	32
Table 3. CRISPR-gRNA sequence	32
Table 4. Primer sequences for PCR	32
Table 5. Antibodies list	33

 
FIGURE DIRECTORY

Figure 1. Effects of hypoxic mimetic reagents, DMOG and CoCl2 on BIG1 and BIG2 expression.	34
Figure 2. mRNA levels of BIG1 and BIG2 under hypoxia.	35
Figure 3. Effects of DNA methylation inhibitor 5’-azacytidine and HDAC inhibitor sodium butyrate treatment under normoxia or hypoxia.	37
Figure 4. Expression of BIG1 and BIG2 in cancer	38
Figure 5. mRNA levels of BIG1 or BIG2 were reduced by infection with shBIG1 or shBIG2 lentivirus	40
Figure 6. Protein abundances of BIG1 or BIG2 were reduced in cells that infection with shBIG1, or shBIG2 lentivirus.	41
Figure 7. Effects of shRNA-mediated BIG1 or BIG depletion on cell morphological change.	42
Figure 8. Cobalt chloride treatment promotes cell migration in transwell migration assay	43
Figure 9. Analysis of cell migration by wound-healing assay	44
Figure 10. Effects of BIG1 and BIG2 depletion on cell viability and proliferation	45
Figure 11. Effects of BIG1 and BIG2 knockout on cell viability and proliferation.	46
Figure 12. Flow cytometric analysis of the cell cycle phase distribution in BIG1- or BIG2-knockdown cells	47
Figure 13. Effects of knockdown of BIG1 and BIG2 on gene expression	48
Figure 14. Effects of knockdown of BIG2 on cell proliferation in vivo	49
Figure 15. Gene structures of BIG1 or BIG2	50
Figure 16. Model of the role of BIG1 and BIG2.	51
Figure 17. Expression of BIG1 and BIG2 in breast invasive carcinoma	52
Figure 18. Expression of BIG1 and BIG2 in cervical squamous cell carcinoma	53
Figure 19. Expression of BIG1 and BIG2 in colon adenocarcinoma	54
Figure 20. Expression of BIG1 and BIG2 in head and neck squamous cell carcinoma	55
Figure 21. Expression of BIG1 and BIG2 in kidney renal clear cell carcinoma	56
Figure 22. Expression of BIG1 and BIG2 in liver hepatocellular carcinoma	57
Figure 23. Expression of BIG1 and BIG2 in lung squamous cell carcinoma	58
Figure 24. Expression of BIG1 and BIG2 in rectum adenocarcinoma	59
Figure 25. Expression of BIG1 and BIG2 in thyroid carcinoma	60


                                    

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