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系統識別號 U0026-1508201415514600
論文名稱(中文) 腫瘤相關巨噬細胞透過正調控微核醣核酸-210促進胃癌腫瘤生成
論文名稱(英文) Tumor-associated macrophages promote gastric cancer tumorigenesis via upregulation of miR-210
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 102
學期 2
出版年 103
研究生(中文) 蔡伊閔
研究生(英文) I-Min Tsai
學號 s96014035
學位類別 碩士
語文別 英文
論文頁數 57頁
口試委員 指導教授-沈延盛
口試委員-呂佩融
口試委員-洪澤民
口試委員-葉大森
中文關鍵字 胃癌  微核醣核酸  腫瘤巨噬細胞 
英文關鍵字 Gastric cancer  MicroRNA  Tumor-associated macrophages (TAM) 
學科別分類
中文摘要 許多臨床研究顯示發炎是促進癌症發生的因素之一,這些浸潤的發炎細胞即是腫瘤巨噬細胞(TAM),腫瘤微環境中腫瘤巨噬細胞會影響腫瘤細胞的生長並促進其轉移。微核醣核酸(microRNA)被發現參與許多生理調控以及疾病的發生其中包括癌症。然而,微核醣核酸及腫瘤巨噬細胞之間詳細的調控機制仍需進一步的研究證實。我們先前的研究顯示腫瘤巨噬細胞存在與否會影響胃癌細胞中微核醣核酸的表現,並發現其中微核醣核酸-210 會有顯著的高表現。因此,在本研究我們認為腫瘤巨噬細胞會調控微核醣核酸-210並促進胃癌轉移可能性。首先,想了解微核醣核酸-210和腫瘤巨噬細胞的關係,我們發現微核醣核酸-210在手術後的臨床胃癌標本以及腫瘤巨噬細胞共培養系統中都有高表現,並在胃癌病人中發現腫瘤巨噬細胞高度浸潤的病人有比較短的總生存時間。此外,微核醣核酸-210去活化會衰減腫瘤巨噬細胞誘導的胃癌細胞轉移。接著,我們利用微核醣核酸資料庫以及MetaCore等分析軟體分析,認為調控細胞骨架的基因NTN4是微核醣核酸-210的標的基因,並發現在胃癌細胞中微核醣核酸-210會抑制含有NTN4 3’ 非編碼區(3’UTR)的冷光報告基因的活性。另外,NTN4的表現量在手術後的臨床胃癌標本以及腫瘤巨噬細胞共培養系統會有減少的趨勢,胃癌病人中NTN4高表現伴隨微核醣核酸-210低的群組有較長總生存時間,NTN4以及微核醣核酸-210可以用來預測病人存活時間。最後,根據以上的結果讓我們了解微核醣核酸-210在發炎系統中所扮演的角色,並發現一個新的訊息路徑:腫瘤巨噬細胞/微核醣核酸-210/NTN4,他們可以作為一個胃癌病人癒後生存時間的生物標誌或是可能的治療靶點。
英文摘要 Chronic inflammation is a crucial event in progression of gastric cancer. Particularly, tumor-associated macrophages (TAMs) are part of inflammatory circuits that promote tumorigenesis. MicroRNAs (miRNAs) are involved in multiple biological activities as well as disease progression including cancer. However, little is known about the association between tumor miRNAs and TAMs. Previously, we compared the expression profile of miRNAs between gastric cancer cells alone and gastric cancer cells co-cultured with U937, and found that miR-210 expression in gastric cancer cells significantly increased after co-cultured. In this study, we sought to determine whether TAMs regulate tumor miR-210, and thus promoting tumorigenesis of gastric cancer. We first verified the correlation between miR-210 and TAMs in gastric cancer and found that miR-210 was up-regulated by TAMs in co-culture experiments. Furthermore, we also observed a positive correlation between miR-210 expression and TAMs in gastric cancer specimens. Overall survival in gastric cancer patients with higher CD204 expression was significantly reduced than patients with lower CD204 expression. In addition, inactivation of miR-210 attenuated TAMs-mediated gastric cancer cell migration. Using MetaCore, we found that NTN4, a regulator for cytoskeleton, is a putative target gene of miR-210. Indeed, miR-210 can block the luciferase activity of NTN4–3’UTR in gastric cancer cell. Moreover, expression of NTN4 was decreased in clinical specimens and gastric cancer cells after TAMs stimulation. Combined high NTN4 expression and lower miR-210 expression is associated with better survival in gastric cancer patients. Collectively, these findings extend our understanding of the function of miR-210 in the inflammation system and this newly identified TAMs/miR-210/NTN4 pathway can be a prognostic factor for gastric cancer patient survival and a potential therapeutic target in gastric cancer treatment.
論文目次 摘要 I
Abstract II
Acknowledgement IV
Contents VI
I.Abbreviation 1
II.Introduction 2
Gastric cancer 2
Tumor microenvironment 3
Tumor-associated macrophages 4
Biogenesis and functions of miRNAs 5
The role of microRNA in cancer 7
III.Materials and Methods 10
Cell culture 10
Co-culture system 10
Total protein extraction 10
Isolation of nuclear and cytoplasmic protein 11
Western blot (WB) 11
RNA extraction 12
Reverse transcription 12
Analysis of the relative amount of NTN4 mRNA by RT-PCR 13
Real-time polymerase chain reaction (qPCR) 13
Quantitative RT-PCR assays for mature miRNA 14
Immunohistochemistry (IHC) 14
In situ hybridization (ISH) 15
Construction of the 3’-UTR-luciferase plasmid and reporter assay 16
Migration assay 17
Oligonucleotide transient transfection 17
Transformation 18
Plasmid isolation 18
Statistical analysis 19
IV.Results 20
MiR-210 expression is upregulated by TAMs in gastric cancer cells. 20
TAMs upregulate miR-210 expression and correspond to decrease overall survival time in gastric cancer patients. 21
Infiltration of tumor-associated macrophages (TAMs) positively correlates with expression of miR-210 in gastric cancer cells. 22
Inhibition of miR-210 attenuates TAMs-induced cell migration. 22
Co-culture of gastric cancer cells with U937 enhances NF-κB and HIF-1α signaling in gastric cancer cells. 23
Infiltration of TAMs increases miR-210 which down-regulates NTN4 expression. 24
MiR-210 regulated NTN4 by directly targeting its 3’-UTR. 25
Inhibition of miR-210 attenuates down-regulation of NTN4. 26
Combined NTN4 and miR-210 expression predict overall survival in gastric cancer patients. 26
Increased miR-210 expression suppresses NTN4 expression in murine tumors. 27
V.Discussion 28
VI.References 32
VII.Figures 38
VIII.Tables 53
Table 1. Top 20 microRNAs down- or over-expression in AGS cell after co-cultured. 53
Table 2. Candidate targets of miR-210 in Gastric cancer 54
IX.Appendix 55
Appendix 1. List of antibody 55
Appendix 2. List of primer 56
X.Curriculum vitae 57
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