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系統識別號 U0026-0708201215340900
論文名稱(中文) 研究hRAD9在大腸癌細胞中促進細胞老化和調節上皮間葉轉化的功能性角色
論文名稱(英文) Investigation on the Functional Role of hRAD9 in Promoting Senescence and Modulating Epithelial-Mesenchymal Transition in Colon Cancer
校院名稱 成功大學
系所名稱(中) 生物科技研究所碩博士班
系所名稱(英) Institute of Biotechnology
學年度 100
學期 2
出版年 101
研究生(中文) 吳姝儀
研究生(英文) Shu-Yi Wu
學號 l66991049
學位類別 碩士
語文別 英文
論文頁數 97頁
口試委員 指導教授-張敏政
口試委員-陳宗嶽
口試委員-黃玲惠
口試委員-洪健睿
口試委員-張純純
中文關鍵字 hRAD9  細胞老化  上皮間葉轉化 
英文關鍵字 hRAD9  senescence  EMT 
學科別分類
中文摘要 在癌症發展過程中,腫瘤是否能維持原位良性瘤或者發展為惡性癌症轉移主要關鍵取決於腫瘤剛新生的時期。在此時期,腫瘤細胞能夠透過特定分子的作用,掙脫細胞凋亡與細胞老化的限制,並誘導上皮間質轉化以增強爬行與侵犯的能力,利於癌症轉移。因此,若強迫癌細胞進入細胞老化,將有機會大幅降低腫瘤生成與癌症轉移的發生率。然而,在目前的研究中,能夠同時參與在促進細胞老化且抑制上皮間葉轉化的分子仍是未知。另外,許多蛋白在癌症發展過程中曾被報導過扮演著雙面性的角色,例如像是細胞週期蛋白D1或是轉化生長因子β,而實驗室所研究的hRAD9蛋白也是其中之一。hRAD9為分裂酵母裡Rad9的同源蛋白,在哺乳動物中為細胞週期檢查點之一,不僅如此,hRAD9在細胞中亦參與了多種不同之生理功能,如去氧核醣核酸修補機制、細胞凋亡、同源重組以及基因轉錄調控等。雖然hRAD9在細胞中參與了許多生理功能,但由於hRAD9扮演著雙面性的角色,此蛋白在癌症發展過程上所扮演的角色至今卻未明朗。先前的研究顯示,在肺癌與乳癌中,hRAD9可能具有致癌性的功能。然而,亦有研究指出,在前列腺癌中,hRAD9能抑制雄性素受器的活化進而抑制癌症進程,且當小鼠的角質細胞缺失RAD9基因時,將導致其表皮易因致癌劑刺激而好發腫瘤。因此,本研究欲探討hRAD9在癌症進程中所扮演的角色與下游之分子機制。首先,在大腸癌的檢體中,我們觀察到hRAD9在惡化的組織中是有較低的表現量。所以,第一步我們則先在較低表現hRAD9的惡化大腸癌細胞SW620中,建立hRAD9蛋白大量表現穩定的細胞株,稱為SW620-hRAD9。實驗結果發現,相較於控制組的細長細胞型態,SW620-hRAD9多呈現較大且攤平的細胞型態。同時,藉由細胞計數與Ki-67染色,證實SW620-hRAD9的細胞增生明顯受到抑制,佐以SA-β-gal與應力纖維的染色等實驗果,充分證實hRAD9的大量表現能夠迫使具有轉移能力的惡性癌細胞再次進入細胞老化。另一方面,我們想知道,是否hRAD9的缺失會導致細胞產生上皮間葉轉化的發生,透過核醣核酸干擾技術在高表現hRAD9的大腸癌細胞HT-29中,抑制hRAD9表現後,經由西方點墨法以及免疫螢光染色確認了在hRAD9表現量被抑制時,間葉細胞標誌有表現量增加的情形而上皮細胞標誌則有表現量減少的情形。最後,也經由博登細胞移形器分析來觀察細胞爬行能力。結果顯示在hRAD9表現量被抑制時,細胞爬行能力有上升的情形。綜合以上結果所得到的結論為hRAD9蛋白同時參與了誘發細胞老化的功能和抑制上皮間葉轉化的功能。而為了使得hRAD9所扮演的抑癌角色有更明確的定位及未來應用於治療癌症的可能性,更多詳細的機制還是需要被探討的。
英文摘要 During cancer progression, neoplasia is the key turning point in determining whether a tumor remains in situ or further progress to the metastatic stage. In neoplasia, tumor cells can act through specific molecules to relieve themselves from the control of senescence or apoptosis, and thus obtain invasive and migratory abilities through the process of epithelial-mesenchymal transition (EMT). Although molecules that force tumor cells to be controlled by senescence are vital in preventing formation of invasive and metastatic tumors, no previous studies have reported a particular molecule with the ability to induce senescence and inhibit EMT simultaneously. In neoplasia, many proteins have been reported with two-sided functions, as either a tumor suppressor or an oncogenic protein, such as Cyclin D1 and TGF-β. Like these proteins, hRAD9, one of the cell cycle checkpoints in humans, is a protein with two-sided functions. The protein RAD9 was first discovered in Schizosaccharomyces pombe; hRAD9 is the human homologue of the yeast protein RAD9. As a cell cycle checkpoint, hRAD9 plays an essential part in various functions, such as in DNA repair, apoptosis, and transcription activity. Although hRAD9 possesses many biological functions, its role in tumorigenicity remains unknown. Some studies have indicated the role of hRAD9 as an oncogenic protein by suggesting that the accumulation of abnormal amounts of hRAD9 protein was found in the cell nuclei of breast and lung cancer tissue. In contrast, hRAD9 appears to function as a tumor suppressor in prostate cancer by interfering with androgen receptor transactivation. RAD9 deletion in mouse keratinocytes has also been shown to promote tumor formation subsequent to carcinogen application. In light of the multiple and contrasting roles attributed to RAD9 by previous studies, the aim of our research is to investigate the role of hRAD9 in tumor progression and the mechanism of downstream effectors. In the first part of our study, lower hRAD9 expression was observed in tumor tissues in colorectal clinical samples. Next, the more malignant colon cell line SW620 with lower hRAD9 expression was utilized to observe the effect of hRAD9 expression on tumor cell behavior. The stable cell line overexpressing hRAD9 was established in SW620 and was named SW620-hRAD9. Compared to its control, SW620-hRAD9 lost fibroblast-like morphology, flattened, and became larger. The cell counting assay, colony formation assay, and Ki-67 marker staining revealed slower cell proliferation in SW620-hRAD9 compared to the control. Since slower cell proliferation and the morphological changes were consistent with the features of senescent cells, functional assays such as senescence-associated β-galactosidase (SA-β-gal) and stress fibers formation were performed to confirm our observations. The results demonstrated that SW620-hRAD9 acquired senescent features compared to the control. From the results, hRAD9 overexpression serves the function of forcing tumor cells with metastatic ability to undergo senescence and prevent further cancer progression. In another part of this study, we explore the effects of depressed hRAD9 on EMT. The less malignant colon cancer cell line HT-29 stable was established through RNAi interference. With Western blot and immunoflourescence analyses, HT-29 shRAD9 (stable hRAD9 knockdown HT-29) exhibited increased expression in the mesenchymal marker vimentin and decreased expression in the epithelial marker E-cadherin. In addition, Boyden chamber utilization showed an increase in cell migration during hRAD9 depression. Overall, hRAD9 is demonstrated to be a tumor suppressor with the ability to induce senescence and inhibit EMT. However, further research is still required to elucidate the mechanisms behind hRAD9 functions and its potential future applications in cancer therapy.
論文目次 中文摘要…………………………………………………………………I
Abstract…………………………………………………………………III
Acknowledgement (誌謝)…………………………………………………V
Table of Contents……………………………………………………VII
List of Tables and Figures……………………………………………X
Abbreviations of Scientific Terms…………………………………XI
Chapter 1 Introduction…………………………………………………1
1-1 Cancer Progression………………………………………………1
1-2 Colorectal Cancer…………………………………………………2
1-3 Introduction of hRAD9……………………………………………4
1-3-1 Functional Domains of hRAD9…………………………………5
1-3-2 Biological Functions of hRAD9………………………………7
1-3-3 Two-side Role of hRAD9 in Cancer Progression…………9
1-4 Discovery and Mechanisms behind Senescence………………10
1-4-1 Cell Cycle………………………………………………………11
1-4-2 Induction Signals of Senescence…………………………13
1-4-3 Molecular Mechanism behind Senescence…………………15
1-5 Epithelial-Mesenchymal Transition (EMT)…………………17
1-6 Purpose of Research……………………………………………19
Chapter 2 Materials and Methods……………………………………20
2-1 Bacterial strain and vectors…………………………………20
I. Bacterial strain…………………………………………………20
II. Vectors……………………………………………………………20
2-2 Gene cloning………………………………………………………20
I. Preparations of Antibiotics…………………………………20
II. Preparations of Media…………………………………………20
III. Plasmid Extraction……………………………………………21
IV. Polymerase Chain Reaction……………………………………22
V. DNA purification…………………………………………………23
VI. Enzyme Restriction……………………………………………24
VII. Ligation…………………………………………………………24
VIII. Preparation of competent cell and transformation …24
2-3 Techniques for cell cultivation……………………………26
I. Cell lines used in laboratory………………………………26
II. Unfreezing cells………………………………………………27
III. Subculture of adherent cells.…………………………….27
IV. Cell counts………………………………………………………28
V. Transfection………………………………………………………29
VI. Preservation of cell lines …………………………………29
2-4 Protein analysis…………………………………………………30
I. Isolation of protein……………………………………………30
II. Quantification of protein……………………………………31
III. SDS-PAGE ………………………………………………………31
IV. Western blot ……………………………………………………33
2-5 Establishment of stables in cancer cell line……………35
2-6 Cell counting assay.……………………………………………36
2-7 Colony formation assay…………………………………………36
2-8 Ki-67 staining for assessment of cell proliferation …37
2-9 Analysis of senescence activity in cell culture………38
2-10 Assay for stress fibers observation………………………39
2-11 Immunoflourescence assay……………………………………40
2-12 Boyden chamber assay…………………………………………41
2-13 Matrigel transwell invasion assay…………………………43
2-14 Statistical Calculation………………………………………44
Chapter 3 Results………………………………………………………45
3-1 hRAD9 expression in tumor tissues…………………………45
3-2 Establishment of hRAD9-overexpressing stable in studying the role of hRAD9 in tumor progression………………46
3-2-1 Observation of morphology change in SW620 stables…47
3-2-2 Assessment of cell growth rate of SW620 stables using cell counting assay……………………………………………………47
3-2-3 Observation of colony-forming ability of SW620 stables……………………………………………………………………48
3-2-4 Utilization of the Ki-67 marker to assess cell proliferation of SW620 stables……………………………………49
3-2-5 Uses of the senescence-associated β-galactosidase (SA-β-gal) assay to observe senescence expression in SW620 stables……………………………………………………………………49
3-2-6 Observation of stress fibers in SW620 stables………50
3-3 Utilize siRNA transient transfection to observe EMT markers expression in HT-29…………………………………………51
3-4 Establish hRAD9-knockdown stable in studying role of hRAD9 in tumor progression…………………………………………52
3-4-1 Observation of morphology change in HT-29 stables…52
3-4-2 Utilize cell counting technique in assessing cell growth rate of HT-29 stables………………………………………53
3-4-3 Observe EMT markers expression in HT-29 stables through Western blot…………………………………………………54
3-4-4 Observe EMT markers expression in HT-29 stables through immunofluorescence…………………………………………55
3-4-5 Use Boyden chamber to observe migratory ability in HT-29 stables…………………………………………………………55
3-4-6 Use Matrigel transwell assay to observe invasive ability in HT-29 cells expressing shLuc or shRAD9…………56
Chapter 4 Conclusion…………………………………………………58
Chapter 5 Discussion…………………………………………………60
5-1 Functional Role of hRAD9 in Cancer Progression…………60
5-2 Effect of hRAD9 Overexpression on Colorectal Cancer Cells………………………………………………………………………61
5-3 Effect of hRAD9 Deprivation on Invasive and Metastatic Properties in Colorectal Cancer Cells……………………………62
5-4 Potential of hRAD9 for cancer therapeutic applications……………………………………………………………64
Figures and Tables……………………………………………………66
References………………………………………………………………81
Appendix…………………………………………………………………92
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