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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 葉秀慧 | |
dc.contributor.author | Po-Jen Chen | en |
dc.contributor.author | 陳柏任 | zh_TW |
dc.date.accessioned | 2021-06-16T17:39:53Z | - |
dc.date.available | 2022-12-31 | |
dc.date.copyright | 2012-09-19 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-08-14 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64307 | - |
dc.description.abstract | microRNA 可能在癌症中扮演類似致癌基因或是腫瘤抑制基因的重要角色, 而在肝癌 (Hepatocellular carcinoma;HCC) 中也已經發現許多表現異常的 microRNA, 但是在肝癌形成的早期過程, 是否就有 microRNA 變異參與在其中卻很少被探討。 本研究利用肝癌鄰近的非腫瘤組織當作癌症前期組織, 藉著 Real-Time PCR 分析 22 個與 HCC 相關的 microRNA 後, 結果發現 miR-216a 和 miR-224 的表現量在癌症前期就開始異常上升, 其中 miR-216a 的上升現象在男性病患尤其顯著。 後續針對 miR-216a 進行研究性別差異的原因, 結果發現雄激素訊息傳遞路徑會促進肝癌細胞內生性的 pri-miR-216a 表現量, 而且此現象在 B 型肝炎病毒的 X 蛋白 (HBx) 存在下會更加明顯。
利用 5’ RACE 找出 pri-miR-216a 5’ 上游可能之轉錄起始位置 (Transcriptional Starting Site;TSS), 針對此 TSS 構築數個包含 TSS 上游不同長度之 reporter 質體, 界定 TSS 上游 –603 到 –304 bp 之基因片段為接受雄激素調控之區域。 進一步利用點突變的實驗確認 –349 到 –335 bp 之間的雄激素反應序列 (androgen response element;ARE) 是 AR 調控轉錄的重要關鍵位置。 之後利用染色質免疫沉澱分析 (Chromatin immunoprecipitation;ChIP) 進一步證實受 ligand 刺激的 AR 的確會與 promoter 上的這個 ARE 作結合, 進而去促進 miR-216a 的轉錄。 除了 miR-216a 的上游調控機轉發現外, 我們也發現 miR-216a 會與 TSLC1 抑癌基因的 3’ 端未轉譯區 (3’ untranslated region;3’ UTR) 結合而降低 TSLC1 蛋白質的表現, 進而去促進肝細胞的增生和遷移之活性。 最後分析臨床上男性病患的肝臟組織, 發現從肝癌前期到演變成肝癌的過程, AR 蛋白表現量會增加, 伴隨著 miR-216a 表現上升及 TSLC1 表現下降, 具顯著相關。 本研究因此指出一雄激素訊息傳遞路徑促進 miR-216a 之轉錄進而降低抑癌基因 TSLC1 表現之調控機轉, 對於肝癌形成早期提供了一個新穎的分子機制。 | zh_TW |
dc.description.abstract | Deregulation of microRNAs (miRNAs) is common in advanced human hepatocellular carcinomas (HCC); however, the ones involved in early carcinogenesis have not yet been investigated. By examining the expression of 22 HCC-related miRNAs between precancerous and cancerous liver tissues, we found miR-216a and miR-224 were significantly upregulated, starting from the precancerous stage. Furthermore, the elevation of miR-216a was mainly identified in male patients. To study this gender difference, we demonstrated that pri-miR-216a is activated transcriptionally by the androgen pathway in a ligand-dependent manner and is further enhanced by the hepatitis B virus X protein. The transcription initiation site for pri-miR-216a was delineated, and one putative androgen responsive element site was identified within its promoter region. Mutation of this site abolished the elevation of pri-miR-216a by the androgen pathway. One target of miR-216a was shown to be the TSLC1 tumor suppressor mRNA through the three target sites at its 3’ UTR. Finally, the androgen receptor level increased in male liver tissues during hepatocarcinogenesis, starting from the precancerous stage, with a concomitant elevation of miR-216a but a decrease of TSLC1. Conclusion: The current study discovered the upregulation of miRNA-216a by the androgen pathway and a subsequent suppression of TSLC1 as a new mechanism for the androgen pathway in early hepatocarcinogenesis. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T17:39:53Z (GMT). No. of bitstreams: 1 ntu-101-D95445006-1.pdf: 4004606 bytes, checksum: 77f46af5495f2be71dee8026e5d88787 (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 口試委員會審定書…………………………………………………………………….I
致謝………………………………………………………………………..…….……II 中文摘要……………………………………………………………………..………III 英文摘要……………………………………………………………………..………IV 第一章 緒論………………………………………………………………..…………1 1.1 肝癌 (Hepatocellular carcinoma;HCC)………………………………….........1 1.2 肝癌之性別差異………………………………………………………….……2 1.3 雄激素受體 (androgen receptor;AR)………………………………….….…3 1.4 微核醣核酸 (microRNA;miRNA)…………………………………………..4 1.5 肝癌與 microRNA 的研究………………………………………………...…5 1.6 抑癌基因 TSLC1 (Tumor suppressor in lung cancer 1)………………………7 1.7 研究目的及策略……………………………………………………………….8 第二章 實驗材料與方法…………………………………………………………..…9 2.1 臨床肝臟組織檢體的選擇及來源…………………………………………….9 2.2 5’ RACE 分析………………………………………………………………..9 2.3 構築 pri-miR-216a 5’ 端 promoter 區域的 reporter 質體…….…………...9 2.4 染色質免疫沉澱分析 (Chromatin immunoprecipitation;ChIP) ……..…….10 2.5 細胞培養、轉染 (transfection) 和冷光酶測定分析 (luciferase reporter assay)…………………………………………………………………………11 2.6 成熟 miRNA 的反轉錄聚合酶連鎖反應定量分析 (Quantitative reverse-transcription PCR)…………………………………………………...12 2.7 西方墨點法 (Western blot analysis)………………………………………....13 2.8 構築 TSLC1 的 reporter 質體和表現質體……………………………..…14 2.9 pri-miR216a 和 TSLC1 mRNA 的定量分析…………………………..…15 2.10 Lentivirus-based microRNA 的構築和製備………………………...……15 2.11 細胞增生 (proliferation) 和遷移 (migration) 試驗……………………...16 2.12 微陣列分析 (Microarray analysis)…………………………………………17 2.13 統計學分析……………………………………………………………….…18 2.14 miR-216a 上升和 HCC 預後之間的相關研究……………………….…19 第三章 實驗結果…………………………………………………………………....20 3.1 在肝癌形成過程中, miR-216a 和 miR-224 的表現量從癌症前期 (precancerous stage) 就開始顯著地增加…………………………………...20 3.2 在肝細胞中 miR-216a 之轉錄會受到雄激素途徑的正向調控, 這可用來解釋 miR-216a 的表現量在男性會更顯著地上升………………………..21 3.3 受到配體 (ligand) 刺激的 AR 可以跟 miR-216a promoter 區域上的 ARE 位置結合進而促進 pri-miR-216a 的轉錄……………………….….22 3.4 miR-216a 表現量上升會使 TSLC1 的表現下降進而去促進肝細胞的 proliferation 和 migration 之活性…………………………………….…24 3.5 在臨床檢體中, miR-216a 表現量上升分別與 AR 蛋白質增加和 TSLC1 蛋白質減少有關聯性……………………………………………………..…26 第四章 結果討論…………………………………………………………………....27 4.1 C 型肝炎病毒相關肝癌 (HCV-related HCC) 病患的非腫瘤肝臟組織所表現 miR-216a 似乎也有性別差異的趨勢…………………………….….…27 4.2 miR-224 目前的相關研究………………………………………………….27 4.3 如何決定 miR-216a 的轉錄起始位置 (Transcriptional Starting Site;TSS)……………………………………………………………………..……28 4.4 在臨床檢體中, TSLC1 mRNA (肝臟腫瘤組織相較於其配對之鄰近非腫瘤部分) 的相對表現程度之探討…………………………………………...…29 4.5 在臨床檢體中, AR、miR-216a、TSLC1 三者之探討………………………29 4.6 與肝癌前期有關的 microRNA 之探討…………………………………….30 4.7 在肝癌形成過程中之 AR 蛋白質的表現變化………………………….…31 4.8 為何在 FNH 的非腫瘤組織部份, miR-216a 的表現沒有性別差異….....32 4.9 miR-216a 下游基因之探討以及與 HCC 預後的關係……………….…..32 4.10 從肝癌前期到肝癌過程中 miR-216a 的表現變化之探討…………….…33 4.11 與 AR 相關的調控機制之探討……………………………………..….…33 4.12 與 TSLC1 相關的調控機制之探討………………………………….……34 4.13 女性的肝癌發生機制之探討………………………………………….……35 4.14 此篇論文的研究貢獻以及未來展望……………………………….……....35 圖表………………………………………………………………………………..…38 圖 1. microRNA 可能參與在男性肝癌形成機制中之假說…………...……..…38 圖 2. microRNA 的生合成與作用以及可能受 AR 的調控………...……...…..39 圖 3. 肝癌形成早期有變化的 microRNA……………………………………...…40 圖 4. 抑癌基因 TSLC1 的結構…………………………………………………...41 圖 5. 在肝癌形成的早期過程中 miR-216a 和 miR-224 的表現量就開始增加.42 圖 6. 受 ligand 刺激的 AR 可以促進 pri-miR-216a 和 miR-216a 的上升, 並且當 HBx 存在下, 上升的現象會更顯著………………………………43 圖 7. 確認 pri-miR-216a 的 TSS 之所在………………………………………..44 圖 8. 受 AR 調控的 pri-miR-216a promoter 區域………………………………45 圖 9. 確認 pri-miR-216a promoter 的 ARE 之所在…………………………….47 圖 10. TSLC1 是 miR-216a 的目標基因………..…………………………...…48 圖 11. miR-216a 調控 TSLC1 所扮演的功能………..……………………...…50 圖 12. 在臨床檢體中, AR 蛋白質表現上升和 TSLC1 蛋白質表現下降都和 miR-216a 表現上升有關聯性……………………………………….……52 圖 13. 肝癌病患中 TSLC1 mRNA 的相對表現程度………………………….…54 圖 14. 肝癌形成過程中之 AR 蛋白質的表現變化…………………...…………55 圖 15. 結論︰ miR-216a 可能參與在男性肝癌形成的機制中……..……...……56 表 1. 抑癌基因 TSLC1 的表現量下降及可能機制……………………………...57 表 2. 臨床檢體之 29 個 microRNA 表現型態之整理………………………….58 表 3. 臨床檢體之相關資料………………………………………………………...59 表 4. 藉由 microarray 結果分析 miR-216a 之可能功能..………..………...…..60 參考文獻……………………………………………………………………………..61 附錄一: 5’ RACE之實驗流程………………………………………………..……71 附錄二: Real-time PCR 偵測 mature microRNA 之原理…………………….…72 附錄三: 與本論文相關之發表文獻………………………………………………...73 | |
dc.language.iso | zh-TW | |
dc.title | 肝癌形成早期雄激素路徑促進微核醣核酸-216a之轉錄進而降低抑癌基因TSLC1表現之研究 | zh_TW |
dc.title | Androgen Pathway Stimulates MicroRNA-216a Transcription to Suppress the Tumor Suppressor in Lung Cancer-1 Gene in Early Hepatocarcinogenesis | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 陳培哲,周玉山,黃宣誠,詹世鵬 | |
dc.subject.keyword | 微核醣核酸-216a,雄激素, | zh_TW |
dc.subject.keyword | microRNA-216a,androgen, | en |
dc.relation.page | 73 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-08-15 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 微生物學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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