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系統識別號 U0026-1608201219140800
論文名稱(中文) 腫瘤巨噬細胞促進胃癌細胞甲基轉移酶表現進而抑制肌動素調節蛋白之表現
論文名稱(英文) Tumor-associated macrophage promotes DNMT1-mediated GSN silencing in gastric cancer cell
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 100
學期 2
出版年 101
研究生(中文) 楊佳龍
研究生(英文) Chia-Lung Yang
學號 S96991041
學位類別 碩士
語文別 英文
論文頁數 67頁
口試委員 指導教授-沈延盛
口試委員-洪澤民
口試委員-顏家瑞
中文關鍵字 胃癌  肌動素調節蛋白  DNA甲基轉移酶  腫瘤巨噬細胞 
英文關鍵字 Gastric cancer  gelsolin  DNA methyltransferase 1 (DNMT1)  Tumor-associated macrophage (TAM) 
學科別分類
中文摘要 在現行胃癌的治療中,癌細胞轉移是治療上所面臨最大的難題之一,因此本研究期望能找出癌細胞轉移的機制進而抑制癌細胞的轉移。肌動素調節蛋白(Gelsolin),在過去的研究當中被指出具有抑制腫瘤細胞轉移之能力,從我們臨床檢體的免疫染色可以看到gelsolin的表現量在胃癌晚期病患相對於早期病患較低。在某些癌症當中gelsolin的基因被發現是受到DNA過度甲基化的抑制而不表現,在實驗中胃癌細胞株給予去甲基藥5-aza可以增加gelsolin的表現,這說明了gelsolin在胃癌的不表現也是受到DNA過度甲基化的影響。DNA的甲基化來自於DNA甲基轉移酶(DNMTs)的作用,其中DNMT1在許多癌症中發現有異常增加,從胃癌檢體的免疫染色則發現DNMT1的表現量剛好與gelsolin相反,早期病患表現較少,晚期病患表現較高,胃癌細胞株的DNMT1基因剔除可以看到gelsolin的表現增加,說明了在胃癌中DNMT1會抑制gelsolin表現。慢性發炎是促進胃癌惡化的重要因素之一,而且已有研究發現在慢性發炎的過程中伴隨著DNA甲基化程度增加的現象,但是從慢性發炎這個微環境因素到胃癌細胞DNA甲基化之間的作用機制尚未被清楚地證明,於是在這個研究當中我們想證明腫瘤巨噬細胞(TAM)這個在慢性發炎中相當重要的角色,是否會異常活化胃癌細胞中的DNA甲基轉移酶(DNMT1)對肌動素調節蛋白的基因過度甲基化使其不表現,進而讓腫瘤細胞更容易產生轉移現象。在臨床檢體中藉由CD204這個TAM的標記做染色,可以發現在腫瘤細胞當中有TAM的浸潤,而從免疫螢光雙染可以看到在有表現DNMT1的腫瘤細胞周圍有TAM的存在。在胃癌細胞株與U937共同培養的實驗中,可以證明胃癌細胞株在U937的作用下會增加DNMT1的表現,進而抑制gelsolin的表現。結果顯示TAM確實是會促進胃癌細胞的DNMT1表現量增加,DNMT1對gelsolin基因過度甲基化,進而使其表現量減少,最終導致癌細胞轉移。
英文摘要 Metastasis is one of the major difficulties in current therapy of gastric cancer. In this study, we expected to discover mechanisms of metastasis in order to raise possibility in disruption of metastasis. Gelsolin, encoded by GSN, is suggested a possible metastasis suppressor which is silenced by hypermethylation in various cancers. Immunohistochemistry staining of clinical specimen demonstrated that gelsolin expression decreased in gastric cancer tissue compared with normal tissue. By treating gastric cell lines with demethylation agent, 5-aza, up-regulation of gelsolin expression was observed which suggestes that aberrant DNA methylation is also involved in gastric cancer. DNA methylation is catalyzed by DNA methyltranseferase family (DNMTs). DNMT1, one of the families, has been discovered up-regulation in many cancers. We found expression of DNMT1 inversely correlated with gelsolin from immunohistochemistry staining and DNMT1 silencing contributed to elevated gelsolin indicates that the major role of DNMT1 in aberrant methylation of gelsolin. Chronic inflammation is a crucial event in progression of gastric cancer, and increasing DNA hypermethylation followed by chronic inflammation has been identified. However, the mechanism within events is still unknown. Here we proposed that tumor-associated macrophage (TAM) in chronic inflammation plays a causal role to activate DNA methyltransferase 1 (DNMT1) in gastric cancer cell, and further methylation of target gene, GSN. Staining of CD204, a marker of TAM, shows the infiltration of TAM in tumor, and DNMT1 expressed tumor cells are surrounded by TAM via immunofluoresence double staining. Coculture gastric cancer cell line with U937 indeed elevated expression of DNMT1, and thus down-regulated expression of gelsolin. Results suggest that tumor-assaociated macrophage up-regulates DNMT1 expression and consequently silenced GSN by hypermethylation.
論文目次 摘要 I
Abstract III
Acknowledgement V
Contents VII
Abbreviation XI
Introduction 1
Epidemiology of gastric cancer 1
Metastasis in cancer progression 2
Role of gelsolin in metastasis 3
Epigenetic regulation: DNA methylation and DNA methyltransferase 4
Chronic inflammation and DNA methylation 5
Role of tumor-associated macrophage (TAM) in inflammation-related cancer 6
Research motive 7
Material and methods 9
Cell culture 9
Treatment of 5-Aza-2'-deoxycytidine (5-aza) 9
Co-culture 9
RNA interence 10
Immunohistochemistry (IHC) and immunofluorescence (IF) 10
Protein extraction 12
Western blotting (WB) 12
RNA extraction 13
Reverse transcriptase-polymerase chain reaction (RT-PCR) 13
Real-time polymerase chain reaction (qPCR) 14
DNA extraction 15
Bisulfite conversion 15
Pyrosequencing 16
Migration assay 17
Cytokine array 17
Statistical analysis 18
Results 19
Gelsolin is down-regulated in clinical gastric cancer patients. 19
Relatively low expression level of gelsolin in human gastric cancer cell lines compared with normal tissue from clinical specimen. 19
Methylation status of CpG island in GSN promoter. 20
Gelsolin expression is silenced via promoter hypermethylation. 20
Up-regulation of DNMT1 in gastric cancer correlates with poor survival. 21
Expression of DNMT1 shows inverse correlation with gelsolin in gastric cancer specimen. 22
DNMT1 mediates transcriptional repression of gelsolin in gastric cancer. 22
Infiltration of tumor-associated macrophage (TAM) positively correlates with expression of DNMT1 in gastric cancer cell. 23
Co-culture system provides an in vitro microenviroment to mimic infiltration of TAM in gastric cancer cell. 24
Up-regulation of DNMT1 and subsequently transcriptional depression of gelsolin in gatric cancer cell lines after co-cultured with U937. 25
TAM mainly down-regulates expression of gelsolin through a DNMT1-dependent mechanism. 25
TAM promotes cell migration through a DNMT1-dependent mechanism. 26
Discussion 27
References 31
Figures 37
Figure 1. Clinical evidence indicated that gelsolin is down-regulated in gastric cancer. 37
Figure 2. Expression level of gelsolin in human gastric cancer cell lines. 38
Figure 3. Methylation status of CpG island in GSN promoter. 39
Figure 4. Restoration of gelsolin by treated with 5’-aza-2’deoxycytidine (5-aza), a demethylation agent, in gastric cancer cell lines indicated that gelsolin is down-regulated by DNA methylation. 41
Figure 5. Clinical evidence indicated that DNMT1 is up-regulated in gastric cancer. 43
Figure 6. Dislocalization of DNMT1 and geloslin in gastric cancer cells. 44
Figure 7. Expression level of DNMT1 in human gastric cancer cell lines. 45
Figure 8. DNMT1 knockdown in two gastric cancer cell lines, AGS and AZ521, contributes to up-regulation of gelsolin in transcription level. 46
Figure 9. Infiltration of tumor-associated macrophage (TAM) in gastric cancer. 48
Figure 10. DNMT1 expressed tumor cells are surrounded by TAM in close juxtaposition. 49

Figure 11. Schema presents a co-culture system that provides an in vitro microenviroment to mimic infiltration of TAM in gastric cancer cell. 50
Figure 12. Up-regulation of M2 marker and cytokines proved that U937 tends to differentiated into M2 type after co-culture with gastric cancer cell lines. 52
Figure 13. Up-regulation of DNMT1 in gatric cancer cell lines, AGS and HR, and subsequently transcriptional depression of gelsolin after co-cultured with U937. 53
Figure 14. U937 inhibits expression of gelsolin through a DNMT1-dependent mechanism. 55
Figure 15. U937 promotes cell migration through a DNMT1-dependent mechanism. 57
Tables 59
Table 1. Grading of gelsolin expression by IHC staining of human gastric cancer patients. 59
Table 2. Grading of DNMT1 expression by IHC staining of human gastric cancer patients. 60
Table3. Grading of CD204 expression by IHC staining of human gastric cancer patients. 61
Appendix 62
Appendix 1. List of antibody 62
Appendix 2. List of primer 64
Appendix 3. Cytokine array of co-culture 65
Curriculum vitae 67
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