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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 郭靜穎 | zh_TW |
| dc.contributor.advisor | CHING-YING KUO | en |
| dc.contributor.author | 雷善婷 | zh_TW |
| dc.contributor.author | Shan-Ting Lei | en |
| dc.date.accessioned | 2023-09-05T16:10:59Z | - |
| dc.date.available | 2023-11-09 | - |
| dc.date.copyright | 2023-09-05 | - |
| dc.date.issued | 2023 | - |
| dc.date.submitted | 2023-08-02 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/89223 | - |
| dc.description.abstract | 癌細胞不受控制的增殖和血管化不足導致腫瘤內長期缺乏氧氣及養分而形成代謝壓力，代謝壓力的產生會使癌細胞生長停滯，甚至可能導致腫瘤中心壞死。然而，在這種壓力環境下，癌細胞也可能進行代謝性重整得以存活甚至轉移。實驗室先前研究發現，在包含MDA-MB-231細胞在內的多種乳癌細胞株中，Early Growth Response 1 (EGR1)的基因轉錄在營養剝奪條件下會被誘導上調，但對於EGR1如何調節癌細胞對代謝壓力適應性的機制仍不暸解。
EGR1作為轉錄因子，可以被多種刺激物壓力信號誘導。其會啟動下游基因轉錄以調節癌細胞增殖、轉移、侵襲和腫瘤血管生成。本研究發現在營養剝奪條件下，EGR1促進了乳癌細胞的生存和遷移能力。此外，我們的原位異種移植腫瘤模型顯示敲低EGR1顯著抑制腫瘤生長，表明EGR1也可以促進腫瘤進展。綜合以上，EGR1在乳癌細胞遭受代謝壓力時具有促進癌細胞生長及侵襲的功能，我們推測在代謝壓力下誘導之EGR1會增強細胞對代謝壓力的抵抗能力。 在葡萄糖剝奪下，我們從西方墨點法觀察到EGR1出現一條分子量約100 kDa的條帶，推測是轉譯後修飾的結果，我們首先鎖定已知的EGR1轉譯後修飾：SUMOylation。我們透過免疫沉澱分析確認了EGR1的SUMOylation，並建立EGR1位點突變的乳癌細胞株。我們發現K272R突變使100 kDa的EGR1條帶消失，而K425R突變則會顯著降低EGR1轉錄活性。然而，EGR1的SUMOylation並非代謝壓力所誘導，於是我們轉向研究另一個已知的EGR1轉譯後修飾：磷酸化。透過去磷酸酶試驗結果顯示代謝壓力下誘導的100 kDa EGR1為磷酸化修飾的產物。 目前我們對於代謝壓力下誘導的EGR1在乳癌中致癌作用的分子機制的暸解仍不足夠，後續我們將繼續研究代謝壓力下轉譯後修飾對於EGR1功能的影響。這將有助於我們暸解乳癌細胞如何適應代謝壓力，並對其產生抗性的原因。 | zh_TW |
| dc.description.abstract | Uncontrolled proliferation and insufficient vascularization of cancer cells result in intratumoral metabolic stress. Therefore, cancer cells must enhance adaptive responses to support cell viability. In our previous study, we observed the induction of Early Growth Response 1 (EGR1) under nutrient-deprived conditions in various breast cancer cell lines, including MDA-MB-231 cells However, the specific role of EGR1 under metabolic stress still remains unclear.
EGR1 is a transcription factor which is often decreased or undetectable in human breast cancer. It plays a critical role in regulating cancer cell proliferation, metastasis, invasion, and tumor angiogenesis. EGR1 can be rapidly induced by various stimuli, such as growth factor, cytokines, mitogens, or several stress signals, including hypoxia, radiation and oxidative stress. In this study, we demonstrated that EGR1 promoted cell viability and migratory ability of MDA-MB-231 cells under nutrient-deprived condition. Moreover, our xenograft model showed that suppressing EGR1 significantly inhibited tumor growth. These results revealed that EGR1 promotes tumor progression in breast cancer, and the induction of EGR1 under metabolic stress may contribute to cellular resistance to metabolic stress. In western blot analysis of EGR1 protein, an additional band at 100 kDa was observed when cells were subjected to glucose depletion, suggesting post-translational modifications (PTMs). Among various PTMs, we first focused on SUMOylation. The results of immunoprecipitation confirmed that EGR1 was modified by SUMO1. To identify the potential SUMOylation sites on EGR1, predictive algorithms were used. We then established EGR1 SUMOylation site-mutated cell lines. We found that the additional EGR1 protein at 100 kDa was absent in K272R mutant, while the K425R mutant exhibited significantly reduced transactivation activity of ERE-Luc. However, the SUMOylation of EGR1 was not induced by metabolic stress. Therefore, our attention shifted to phosphorylation. In vitro dephosphorylation assay revealed that EGR1 undergoes phosphorylation under glucose, arginine or glutamine deprivation. The molecular mechanisms underlying the tumor-promoting effect of EGR1 in breast cancer under metabolic stress remains unknown. Further investigations are necessary to clarify the role of EGR1 and the various types of PTMs it undergoes. We are eager to understand how these PTMs affect EGR1 transcriptional activation and subsequently regulate EGR1 functions in response to metabolic stress. | en |
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| dc.description.provenance | Made available in DSpace on 2023-09-05T16:10:59Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 誌謝 i
摘要 ii Abstract iii 縮寫表 v 圖目錄 x 附圖目錄 xi 第一章、前言 1 1. 1 乳癌 1 1. 2 腫瘤內代謝壓力 4 1. 3 轉譯後修飾 6 1.3.1 小泛素修飾 7 1.3.2 磷酸化 9 1. 4 早期生長反應家族(Early Growth Response Family) 10 1. 5 Early Growth Response 1 10 1.5.1 EGR1的介紹 10 1.5.2 EGR1的誘導機制 11 1.5.3 EGR1與癌症 12 1.5.4 EGR1與乳癌 12 1.5.5 EGR1的轉譯後修飾 13 第二章、研究目的 14 第三章、研究方法 15 3. 1 細胞培養 15 3. 2 細胞轉染 16 3. 3 RNA基因干擾 16 3.3.1 siRNA轉染試驗 16 3.3.2 shRNA轉染試驗 16 3. 4 慢病毒轉導與篩選 16 3. 5 蛋白質萃取及定量 17 3. 6 西方墨點法 18 3. 7 細胞核細胞質分離 19 3. 8 酸性磷酸酶試驗(Acid phosphatase test) 19 3. 9 Transwell細胞遷移試驗 19 3. 10 海馬生物能量分析XF Cell Mito Stress Test 20 3. 11 質體建構及製備 20 3.11.1 pCMV-Tag2A-EGR1突變株 20 3.11.2 N174-MCS-flag-EGR1突變株 22 3. 12 免疫螢光染色 23 3. 13 免疫沈澱法 23 3. 14 體外去磷酸化試驗 (In vitro dephosphorylation assay) 24 3. 15 螢光素酶報導基因分析 24 3. 16 異種移植試驗 25 3. 17 腫瘤組織蛋白質萃取 25 3. 18 統計分析 25 第四章、結果 26 4. 1 EGR1在代謝壓力下被誘導並分佈於細胞核中 26 4. 2 EGR1在代謝壓力下促進乳癌細胞存活 26 4. 3 EGR1促進乳癌細胞遷移能力 26 4. 4 EGR1抑制乳癌細胞進行粒線體氧化磷酸化 26 4. 5 EGR1促進乳癌細胞腫瘤發展 27 4. 6 EGR1在代謝壓力下會被SUMO修飾 27 4. 7 EGR1 SUMO1修飾不影響蛋白質降解 28 4. 8 利用生物資訊分析平台預測EGR1 SUMOylation之位點 28 4. 9 建立EGR1 SUMOylation位點突變之細胞株 29 4. 10 K272是SUMO1修飾EGR1的位點 29 4. 11 EGR1 SUMO修飾位點突變不影響其分子量大小及其定位 30 4. 12 EGR1 K272位點突變不影響其轉錄活性 30 4. 13 EGR1在代謝壓力下進行磷酸化 31 4. 14 ROS與p38調控EGR1表現量 31 4. 15 GCN2調控EGR1磷酸化 31 第五章、結論與討論 33 圖 39 參考文獻 56 附錄圖 67 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 乳癌 | zh_TW |
| dc.subject | 磷酸化 | zh_TW |
| dc.subject | 小泛素化 | zh_TW |
| dc.subject | 轉譯後修飾 | zh_TW |
| dc.subject | EGR1 | zh_TW |
| dc.subject | 代謝壓力 | zh_TW |
| dc.subject | breast cancer | en |
| dc.subject | metabolic stress | en |
| dc.subject | EGR1 | en |
| dc.subject | post-transcriptional modification | en |
| dc.subject | SUMOylation | en |
| dc.subject | phosphorylation | en |
| dc.title | 探討在乳癌代謝壓力反應中EGR1的轉譯後修飾對其功能之影響 | zh_TW |
| dc.title | Functional Characterization of Post-Translational Modifications on Early Growth Response 1 (EGR1) in Breast Cancer Cells under Metabolic Stress | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 111-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 林亮音;蘇剛毅;楊雅倩;卓爾婕 | zh_TW |
| dc.contributor.oralexamcommittee | LIANG-IN LIN;KANG-YI SU;YA-CHIEN YANG;ER-CHIEH CHO | en |
| dc.subject.keyword | 乳癌,代謝壓力,EGR1,轉譯後修飾,小泛素化,磷酸化, | zh_TW |
| dc.subject.keyword | breast cancer,metabolic stress,EGR1,post-transcriptional modification,SUMOylation,phosphorylation, | en |
| dc.relation.page | 69 | - |
| dc.identifier.doi | 10.6342/NTU202302228 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2023-08-02 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學系 | - |
| dc.date.embargo-lift | 2028-08-01 | - |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
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