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系統識別號 U0026-0508201617132900
論文名稱(中文) 長鏈非編碼核糖核酸ZNF469-3透過調節微小核糖核酸574-5p與ZEB1蛋白之表現量促進三陰性乳癌肺轉移
論文名稱(英文) Linc-ZNF469-3 enhances lung metastasis through regulating miR-574-5p/ZEB1 in triple negative breast cancer
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 104
學期 2
出版年 105
研究生(中文) 周承翰
研究生(英文) Cheng-Han Chou
學號 S96034035
學位類別 碩士
語文別 英文
論文頁數 116頁
口試委員 指導教授-呂佩融
召集委員-洪建中
口試委員-陳芃潔
口試委員-沈湯龍
中文關鍵字 長鏈非編碼核糖核酸ZNF469-3  微小核糖核酸574-5p  ZEB1  肺轉移  乳癌 
英文關鍵字 linc-ZNF469-3  miR-574-5p  ZEB1  lung metastasis  breast cancer 
學科別分類
中文摘要 癌症病患發生遠端器官轉移往往是造成死亡主要原因。於乳癌患者中常見轉移器官為肺、肝、腦以及骨骼,但其專一性組織轉移特性詳細機制仍未知。長鏈非編碼核糖核酸定義為一新類別核糖核酸,其轉錄物長度長於200核甘酸且缺乏轉譯蛋白能力,近期發現能夠調控腫瘤進展過程。在此假設長鏈非編碼核糖核酸參與乳癌的肺臟轉移特性。根據次世代定序結果,於肺組織專一性轉移乳癌細胞株中,長鏈非編碼核糖核酸ZNF469-3被鑑定為與肺轉移特性相關。於細胞實驗結果顯示,過度表現長鏈非編碼核糖核酸ZNF469-3可提升ZEB1蛋白表現進而促進轉移能力。此外,於異種移置小鼠模式動物中,穩定表達長鏈非編碼核糖核酸ZNF469-3細胞株較於對照組具有增加肺轉移能力特性。長鏈非編碼核糖核酸ZNF469-3功能藉由吸附其對應之微小核糖核酸574-5p,阻止微小核糖核酸574-5p影響ZEB1表現量降低。於三陰性乳癌病患中,單獨高表現長鏈非編碼核糖核酸ZNF469-3或合併高表現ZEB1統計結果,與癌症肺轉移復發具相關性。因此,於本研究結果顯示,長鏈非編碼核糖核酸ZNF469-3藉由透過微小核糖核酸574-5p-ZEB1 訊息傳遞路徑,進而增加乳癌肺轉移能力。綜合上述結果,長鏈非編碼核糖核酸ZNF469-3可做為三陰性乳癌肺轉移復發病患診斷及預後指標。
英文摘要 Distant metastasis is a leading cause of death in cancers. The lung, liver, brain, and bone are frequent metastatic sites of breast cancer. The underlying mechanism of breast cancer cells with tissue specific tropism remains unclear. Long noncoding RNAs are defined as transcripts longer than 200 nucleotides that lack protein coding potential and enable to regulate tumor progression. We hypothesized that long noncoding RNAs can mediate the lung tropism in breast cancer. According to Next Generation Sequencing (NGS) data, linc-ZNF469-3 was identified as a lung tropism related lncRNA from lung specific metastatic breast cancer cells. Our results showed that overexpression of linc-ZNF469-3 can increase ZEB1 protein level and metastatic ability in vitro. In addition, linc-ZNF469-3 stably expressing cells can increase lung metastasis ability compared with control group in xenograft mice model. MiR-574-5p was then identified as linc-ZNF469-3 targeting miRNA. We demonstrated that linc-ZNF469-3 may function as a sponge for sequestering miR-574-5p from targeting and down-regulating ZEB1. Furthermore, high linc-ZNF469-3 alone or combined with high ZEB1 is correlated with lung recurrence in triple negative breast cancer patients. The conclusion of the current study is that linc-ZNF469-3 increases lung metastasis ability in breast cancer may through mediating miR-574-5p-ZEB1 pathway. Taken together, our results demonstrate that linc-ZNF469-3 can serve as potential diagnostic and prognostic markers for TNBC lung recurrence patients.
論文目次 Abstract I
中文摘要 II
誌謝 III
Content IV
Abbreviations 1
Chapter 1. Introduction 2
1.1 Breast cancer 2
1.2 Metastasis 3
1.3 Long non-coding RNA 4
1.4 LncRNAs as key regulator in cancer metastasis 5
Chapter 2. Materials and Methods 7
Western blot analysis 7
Cell lines culture 8
RNA extraction 8
cDNA synthesis 9
Quantitative real-time polymerase chain reaction (qRT-PCR) 9
Migration and invasion assays 10
Cell proliferation assay 10
Soft-agar assay 11
MS2-tagged RNA affinity purification (MS2-TRAP) 11
Reporter assay 13
Lentiviral Production 14
Clinical specimens 14
Immunohistochemistry (IHC) 15
In situ hybridization (ISH) 16
Animal studies 17
Statistical analysis 17
Chapter 3. Results 18
High linc-ZNF469-3 is correlated with lung metastasis in triple negative breast cancer. 18
Linc-ZNF469-3 can promote cell metastasis ability in vivo and in vitro. 20
Linc-ZNF469-3 mediated cancer stemness properties 22
Overexpression linc-ZNF469-3 enhances ZEB1 upregulation. 23
MiR-574-5p physically interacts with linc-ZNF469-3. 25
MiR-574-5p up-regulated expression suppressed ZEB1 protein expression is correlated with linc-ZNF469-3 down-regulated and suppressed invasion ability. 26
High linc-ZNF469-3 combined with high ZEB1 is correlated with recurrence in triple negative breast cancer patients. 28
Chapter 4. Conclusion and Discussion 30
Figure and Table 34
References 92
Appendix 100

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