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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 郭彥彬(YEN-PING KUO) | |
dc.contributor.advisor | 郭彥彬(YEN-PING KUO | oddie@ntu.edu.tw | ), | |
dc.contributor.author | You-Yu Liao | en |
dc.contributor.author | 廖友瑜 | zh_TW |
dc.date.accessioned | 2023-03-19T21:18:40Z | - |
dc.date.copyright | 2022-10-03 | |
dc.date.issued | 2022 | |
dc.date.submitted | 2022-08-01 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/83803 | - |
dc.description.abstract | 根據衛福部2020年十大癌症統計口腔癌排名第六名,且近年病患有年輕化的趨勢。其中檳榔內含的檳榔鹼 (Arecoline) 被歸類一級致癌物,亦為口腔癌主要致病因子,但其致癌機轉尚未清楚。過去研究指出,Ras-responsive element binding protein 1 (RREB1) 參與MAPK訊息傳遞路徑,在前列腺癌、結直腸癌、泌尿癌扮演重要的作用。但RREB1在口腔癌癌化與癌幹細胞特性上尚未被探討。先前實驗室發現RREB1在口腔癌OSCC患者檢體中有過度表現,且RREB1蛋白於SAS細胞聚球體中表現量增加。本研究發現以檳榔鹼arecoline刺激Ca9-22與SAS細胞株可誘導RREB1表現,加入TGF-?1中和抗體、ALK5抑制劑 (SB431542)、Smad3抑制劑 (SIS3)可降低由檳榔鹼所誘導的RREB1表現。當加入EGFR抑制劑 (AG1478)、MMP抑制劑 (GM6001)、p53抑制劑(Pifithrin-α)、、p38抑制劑 (SB203580) 、MEK1/2抑制劑 (PD98059) 、JNK抑制劑 (SP600125),可降低由TGF-?1所誘導的RREB1的蛋白表現。以RREB1 siRNA處理RREB1表現量較高的SAS細胞,會降低其Migration與Invasion能力,亦下調由TGF-?1誘導的EMT 相關標誌蛋白。同時RREB1 siRNA可以降低SAS聚球體細胞的形成,並發現CD133、CD44、KLF4、OCT4A、SOX2、Nanog等癌幹細胞標誌表現下降。經過RREB1 overexpression之細胞其proliferation、Migration與Invasion的能力上升,且影響EMT markers,上調N-cadherin、Vimentin、Slug、Snail、Twist表現,下調E-cadherin表現。同時增加聚球體細胞形成並上調CD133、CD44、KLF4、OCT4A、SOX2、Nanog。使用Nod-scid老鼠進行實驗發現RREB1可以促進腫瘤的生長。初步觀察到RREB1 overexpression之細胞會上調p-EGFR、IFIT1、IFIT3的表現,以siIFIT1與siIFIT3處理並降低了RREB1 overexpression細胞的Migration、Invasion能力,下調EMT與癌幹細胞標誌蛋白,同時降低聚球體細胞數量的形成。另以Gefitinib處理72小時RREB1 overexpression細胞後,發現相比Control組具有抑制的效果,顯示Gefitinib藥物具有治療RREB1過表現之口腔癌的潛力。 | zh_TW |
dc.description.abstract | According to the death statistics of Ministry of Health and Welfare from Taiwan in 2020, oral cancer was ranked 6th of the top ten cancer. Furthermore, patients who had oral cancer were getting younger recently. Arecoline in betel nut is classified as a first-class carcinogen, and it was also the main factor of oral cancer. However, its detailed carcinogenic mechanism is not clear yet. Based on past studies, Ras-responsive element binding protein 1 (RREB1) was involved in the MAPK signaling pathway, and played an important role in prostate cancer, colorectal cancer, and urinary cancer when its expression was unbalanced. Nevertheless, the mechanisms of RREB1 in oral cancer carcinogenesis and cancer stem cells were not been full explored. Previously, our laboratory found that RREB1 was overexpressed in oral cancer OSCC patients, and the expression of RREB1 protein was significantly increased in SAS sphere cells. In this study, arecoline was used to stimulate Ca9-22 and SAS cell lines to induce RREB1 expression, and the additions of TGF-?1 neutralizing antibody, ALK5 inhibitor (SB431542), and Smad3 inhibitor (SIS3) could decrease the expression of RREB1 induced by arecoline. When treating EGFR inhibitor (AG1478), MMP inhibitor (GM6001), p53 inhibitor (Pifithrin-α), MEK1/2 inhibitor (PD98059), p38 inhibitor (SB203580), JNK inhibitor (SP600125), it decreased the protein expression of RREB1 induced by TGF-?1. Treating SAS cells with RREB1 siRNA, it reduced the ability of cell migration, invasion, and also down-regulated EMT-related markers which induced by TGF-?1. Moreover, RREB1 siRNA could reduce the formation of SAS sphere cells, and we found that the expression of CD133, CD44, KLF4, OCT4A, SOX2, Nanog decreased. With RREB1 overexpression in cells , the proliferation of cells increased, and the abilities of migration and invasion were also enhanced, and the expressions of EMT markers like N-cadherin, Vimentin, Slug, Snail, Twist were up-regulated , and the expression of E-cadherin was down-regulated. It also increased the formation of sphere cells and up-regulates stemness marker expressions like CD133, CD44, KLF4, OCT4A, SOX2, Nanog.Moreover, We use Nod-scid mice to test and found that RREB1 can promote tumor growth in vivo.In preliminary experiments ,we observed that RREB1 overexpression of cells may up-regulated the expression of p-EGFR, IFIT1, and IFIT3. When treating cells with siIFIT1 and siIFIT3, it might reduced the migration, invasion, the formation of sphere cells, and down-regulated EMT markers and stemness markers of RREB1 overexpressed cells. In addition, it was found to have an inhibitory effect compared with control group when treating the RREB1 overexpression cells with Gefitinib for 72 hours, indicating that Gefitinib might had potential to be a therapeutic drug with RREB1 overexpressed oral cancer. | en |
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dc.description.tableofcontents | 口試委員會審定書 I 誌謝 II 中文摘要 III Abstract IV 目 錄 V 一、研究背景與文獻回顧 1 1.1口腔癌 1 1.2檳榔鹼 3 1.3乙型轉化生長因子TGF-? ( Transforming growth factor ? ) 5 1.4 Ras Responsive Element Binding Protein 1 (RREB1) 7 1.5 Interferon-induced protein with tetratricopeptide repeats 9 二、研究動機與目的 10 三、實驗材料與方法 11 3.1實驗材料 11 3.2 實驗方法 12 3.2.1細胞培養 12 3.2.2細胞繼代培養 12 3.3 藥物處理 13 3.3.1 Seeding cells以及starvation 13 3.3.2 抑制劑、中和抗體 13 3.4 西方墨點法 14 3.4.1蛋白質萃取與定量 14 3.4.2膠體配置 14 3.4.3 蛋白質電泳與轉漬 (Transfer) 15 3.4.4 一級抗體與二級抗體使用 15 3.4.5 顯影呈色 16 3.5 細胞株RREB1基因Knockdown實驗 16 3.6細胞增生速率測試 (Cell proliferation assay) 17 3.7細胞存活率試驗 (MTT assay) 17 3.8細胞移動性實驗 (Migration assay) 17 3.9 細胞侵襲性試驗 (Invasion assay) 18 3.10 細胞聚球體形成試驗 (Sphere forming assay) 18 3.11 細胞聚球體RREB1基因Knockdown實驗 19 3.12 抽取質體與建立RREB1過表現Ca9-22細胞 19 3.13 腫瘤移植實驗 20 四、實驗結果 21 4.1 檳榔鹼誘導Ca9-22細胞與SAS細胞的RREB1蛋白表現 21 4.2 檳榔鹼經由TGF-?路徑誘導Ca9-22細胞與SAS細胞的RREB1蛋白表現 21 4.3 TGF-?1誘導Ca9-22細胞與SAS細胞的RREB1蛋白表現 22 4.4 TGF-?1透過TACE/ADAM17活化EGFR並啟動下游路徑 (MAPK、JNK、p38)誘導Ca9-22細胞與SAS細胞的RREB1蛋白表現。 22 4.5 Knockdown RREB1可降低SAS細胞migration與invasion能力 23 4.6 Knockdown RREB1降低TGF-?1 induced的EMT markers表現 23 4.7 SAS細胞聚球體中RREB1蛋白表現量提高 23 4.8 Knockdown RREB1可降低SAS形成聚球體的能力與癌幹細胞markers 24 4.9 RREB1 overexpression增強Ca-922與TW2.6細胞的生長速度 24 4.10 RREB1 overexpression增強細胞migration與invasion能力 25 4.11 RREB1 overexpression增強EMT markers的表現 25 4.12 RREB1 overexpression增強細胞形成聚球體的能力 25 4.13 RREB1 overexpression增強Stemness markers的表現 26 4.14 RREB1 overexpression促進腫瘤的生長 26 4.15 RREB1 overexpression增強p-EGFR、IFIT1、IFIT3的表現 26 4.16 將RREB1 overexpression之Ca9-22與TW2.6細胞knockdown IFIT1與IFIT3後會降低細胞migration與invasion能力 27 4.17 將RREB1 overexpression之Ca9-22與TW2.6細胞knockdown IFIT1與IFIT3會降低細胞EMT markers表現 27 4.18 將RREB1 overexpression之Ca9-22與TW2.6細胞knockdown IFIT1與IFIT3會降低細胞聚球體的形成 28 4.19 將RREB1 overexpression之Ca9-22與TW2.6細胞knockdown IFIT1與IFIT3會降低細胞EMT與Stemness markers表現 28 4.20 Gefitinib對於RREB1 overexpression之 Ca9-22 與TW2.6細胞具有抑制效果 28 五、討論與結論 30 六、圖與表 33 Fig,1 Arecoline誘導SAS、Ca9-22細胞RREB1蛋白表現 33 Fig.2 Arecoline經由TGF-?路徑誘導SAS及Ca9-22細胞RREB1蛋白表現 34 Fig.3 TGF-?1誘導SAS以及Ca9-22細胞RREB1蛋白表現 35 Fig.4 TGF-?1透過TACE/ADAM17活化EGFR誘導Ca9-22細胞與SAS細胞的RREB1蛋白表現。 36 Fig.5 TGF-?1經MAPK、p38、JNK路徑誘導Ca9-22細胞與SAS細胞的RREB1蛋白表現 37 Fig.6 內生性RREB1在各口腔癌細胞株cell line的表現量 38 Fig.7透過Migration assay分析knockdown RREB1對SAS細胞移動能力的影響 39 Fig.8透過Invasion assay分析knockdown RREB1對SAS細胞侵襲能力的影響 40 Fig.9 Knockdown RREB1對TGF-?1 induced的EMT markers表現影響 41 Fig.10 SAS細胞聚球體中RREB1蛋白表現量 42 Fig.11 Sphere forming assay觀察SAS sphere處理siRREB1後形成聚球體的能力 43 Fig.12分析SAS sphere cells處理siRREB1癌幹細胞相關蛋白的表現 44 Fig.13建立pSPORT6-RREB1 overexpression細胞 45 Fig.14 RREB1 overexpression對細胞生長速率的影響 46 Fig.15透過Migration assay分析RREB1 overexpression對Ca9-22與TW2.6細胞移動能力的影響 47 Fig.16透過Invasion assay分析RREB1 overexpression對Ca9-22與TW2.6細胞侵襲能力的影響 48 Fig.17 Ca9-22與TW2.6細胞wild type、pcDNA3.1及pSPORT6-RREB1之EMT相關標誌蛋白表現 50 Fig.18 Sphere forming assay觀察p-SPORT6 RREB1 Ca9-22形成聚球體的能力 51 Fig.19 Ca9-22與TW2.6 wild type、pcDNA3.1及pSPORT6-RREB1細胞之癌幹細胞相關標誌蛋白表現 53 Fig.20 比較RREB1 overexpression之TW2.6細胞對於腫瘤生長影響 54 Fig.21 Wild type、pcDNA3.1及pSPORT6-RREB1之Ca9-22與TW2.6細胞中p-EGFR、IFIT1、IFIT3蛋白表現 55 Fig.22分析Ca9-22與TW2.6處理TGF-?加入siRREB1後p-EGFR蛋白表現 56 Fig.23透過Migration assay分析對pSPORT6-RREB1 Ca9-22與TW2.6細胞處理siIFIT1與siIFIT3移動能力的影響 57 Fig.24透過Invasion assay分析對pSPORT6-RREB1 Ca9-22與TW2.6細胞處理siIFIT1與siIFIT3移動能力的影響 58 Fig.25分析pSPORT6-RREB1 Ca9-22處理siIFIT1與siIFIT3後EMT相關標誌蛋白表現 60 Fig.26 Sphere forming assay觀察pSPORT6-RREB1 Ca9-22與TW2.6處理siIFIT1與siIFIT3後形成聚球體的能力 61 Fig.27分析RREB1過表現之Ca9-22與 TW2.6處理siIFIT1與siIFIT3後Stemness相關標誌蛋白表現 63 Fig.28分析藥物Gefitinib對於RREB1 overexpression之 Ca9-22與TW2.6 細胞的抑制效果 64 七、參考文獻 65 | |
dc.language.iso | zh-TW | |
dc.title | 檳榔鹼誘導RREB1表現之訊息傳遞路徑與RREB1在口腔癌變的作用 | zh_TW |
dc.title | Signaling pathway of arecoline-induced RREB1 expression and the role of RREB1 in oral carcinogenesis | en |
dc.type | Thesis | |
dc.date.schoolyear | 110-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄭世榮(SHIH-JUNG CHENG),張瑞青(JENNY ZWEI-CHIENG CHANG) | |
dc.subject.keyword | 口腔癌,檳榔鹼,TGF-?,RREB1,癌幹細胞,Gefitinib, | zh_TW |
dc.subject.keyword | Oral cancer,Arecoline,TGF-?1,RREB1,Cancer stem cells,Gefitinib, | en |
dc.relation.page | 71 | |
dc.identifier.doi | 10.6342/NTU202201461 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2022-08-01 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 口腔生物科學研究所 | zh_TW |
顯示於系所單位: | 口腔生物科學研究所 |
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