肝癌之發生具強烈性別特異性，男女比例約為5-7:1。流病學調查顯示此差異可能部分來自於雌激素對女性肝癌之保護作用。過去我們已發現女性肝癌中成熟微型核醣核酸-18a有顯著上升，微型核醣核酸-18a可藉負向調控雌激素受體α，進而抑制女性肝癌之發生。此研究之目的為研究女性肝癌中導致成熟微型核醣核酸-18a表現上升之機制。首先，我們於臨床女性肝癌檢體中觀察到次級微型核醣核酸-18a與成熟微型核醣核酸-18a之表現量具正相關性，但此正相關性不存在於初級微型核醣核酸-18a與成熟微型核醣核酸-18a之表現情形。此結果顯示成熟微型核醣核酸-18a之表現量上升可能源自於微型核醣核酸-18a生成過程中，由microprocessor complex進行之將初級微型核醣核酸-18a裁切成為次級核醣核酸-18a之步驟。Microprocessor complex作用之效率及專一性可受多種細胞內因子影響，其中已知p53蛋白可藉協助穩定microprocessor complex而促使微型核醣核酸-16-1、-143、-145之生成。因此，我們提出p53蛋白可能在女性肝癌中扮演促進微型核醣核酸-18a生成之角色之假說。我們使用細胞培養系統進行實驗以驗證此假說，當細胞經轉染大量表現野生型或突變型p53質體時，成熟微型核醣核酸-18a表現量上升。而當以siRNA降低p53蛋白表現量時，成熟微型核醣核酸-18a之表現量亦下降。上述結果顯示p53蛋白於成熟微型核醣核酸-18a生成路徑中具正向調控功能。此外，我們在女性肝癌臨床檢體中發現成熟微型核醣核酸-18a之表現量上升與p53蛋白之累積具正相關性亦支持此假說。本研究之新穎性在於發現p53蛋白參與女性肝癌中成熟微型核醣核酸-18a之上升，詳細之分子機制仍待進一步闡述。

Hepatocellular carcinoma (HCC) has strong gender disparity that it occurs mainly in men. The male to female ratio is 5-7 to 1. Epidemiologic data shows the differences may partly come from the protection effect of estrogen. Previously, we found that miR-18a elevates significantly in female HCC. And miR-18a can promote female HCC by downregulating its target gene, the estrogen receptor α (ER α). The goal of current research is to investigate the mechanism underlying the elevation of miR-18a in female HCC. We first identified a positive correlation between the level of pre-miR-18a and mature miR-18a in female HCC, which is not exist between the level of pri-miR-18a and mature miR-18a. It thus raised one possibility that the elevation of miR-18a in female HCC may be resulted by enhanced processing of pri-miRNA-18a to pre-miRNA-18a, a step of biogenesis conducted by a microprocessor complex. The efficiency and specificity of microprocessor complex can be regulated by many cellular factors, including p53, which can help stabilize the microprocessor complex and thus enhance the processing of a subgroup of miRNAs from pri- to pre- form. Therefore, we took p53 as a potential candidate for investigating its involvement in the elevation of miR-18a in female HCC. Using the cell culture assay system, miR-18a could be elevated by overexpression of either wild type or mutant p53 constructs. On the other hand, knocking down of p53 by siRNA decreased the miR-18a level. The results thus implicated that p53 could act as a positive regulator in miR-18a biogenesis. Such a finding was further supported by a positive correlation between miR-18a elevation and p53 accumulation preliminarily identified in clinical female HCC samples. Our study thus pointed out a novel mechanism for the involvement of p53 in accelerating the biogenesis of miR-18a in female HCC, with the detail mechanisms awaits further investigation.

口試委員會審定書-i
中文摘要-ii
英文摘要-iii
第一章 研究背景-1
1.1 肝癌 (Hepatocellular carcinoma, HCC)-1
1.2 肝癌之性別差異-1
1.3 微型核醣核酸 (microRNA, miRNA)-2
1.4 癌症中的微型核醣核酸失調-2
1.5 微型核醣核酸的生成-3
1.6 初級微型核醣核酸經processing為次級微型核醣核酸-3
1.7 miR-17-92-4
1.8 miR-18a與肝癌之性別差異-5
1.9 已知之miR-18a processing層面之調控：hnRNPA1-5
1.10 肝癌中之p53蛋白-6
1.11 p53蛋白與微型核醣核酸-6
第二章 研究目的-8
第三章 實驗材料與方法-9
3.1 細胞培養 (Cell culture)-9
3.2 質體轉型 (Transformation)-9
3.3 質體之製備-9
3.4 轉染 (Transfection)-10
3.5 細胞均質液之製備-10
3.6 組織均質液之製備-11
3.7 蛋白質定量-11
3.8 SDS-PAGE-11
3.9 西方墨點法 (Western blot)-12
3.10 抽取RNA-13
3.11 DNase I 處理-13
3.12 將RNA反轉錄為cDNA-14
3.13 反轉錄定量聚合酵素連鎖反應(qRT-PCR)-14
3.14 微型核醣核酸之反轉錄定量聚合酵素連鎖反應 (qRT-PCR)-15
3.15 帶有shRNA之慢病毒顆粒製備-16
3.16 病毒感染-17
3.17 野生型p53質體點突變 (site-directed mutagenesis)-17
3.18 p53定序-18
第四章 實驗結果-21
4.1 女性肝癌具有專一性調控微型核醣核酸-18a上升之機制-21
4.2 成熟微型核醣核酸-18a之上升來自於processing的增加-22
4.3 野生型p53蛋白可提高初級微型核醣核酸-18a之表現量-22
4.4 突變型p53蛋白亦可促進成熟微型核醣核酸-18a之表現-23
4.5 p53蛋白表現上升與微型核醣核酸-18a表現增加同時發生於女性肝癌檢體-24
4.6 微型核醣核酸-18a之表現及p53蛋白之累積相關性較易發現於HBV
陽性之女性肝癌-25
第五章 討論-27
參考文獻-31
附錄-36
圖一(a) 成熟微型核醣核酸-17, 18a, 19a, 20, 19b, 92於女性肝癌中之表現(組別: 成熟微型核醣核酸-18a於腫瘤中上升小於三倍, N=4)-36
圖一(b) 成熟微型核醣核酸-17, 18a, 19a, 20, 19b, 92於女性肝癌中之表現(組別: 成熟微型核醣核酸-18a於腫瘤中上升大於三倍, N=6)-37
圖二 初級微型核醣核酸-18a於女性肝癌中之表現量-38
圖三 次級微型核醣核酸-18a於女性肝炎中之表現量-39
圖四(a) 轉染使野生型p53蛋白表現量上升-40
圖四(b) 野生型p53蛋白轉染對A549細胞之微型核醣核酸-18a表現量影響-40
圖四(c) 野生型p53蛋白轉染對A549細胞之微型核醣核酸-18a表現量影響(以vector控制組標準化後之結果)-41
圖五(a) 以慢病毒系統送入sip53使p53蛋白表現量下降-42
圖五(b) 以慢病毒系統降低p53蛋白表現量對A549細胞之微型核醣核酸-18a表現量影響-42
圖五(c) 以慢病毒系統降低p53蛋白表現量對A549細胞之微型核醣核酸-18a表現量影響 (以vector控制組標準化後之結果)-43
圖六 突變型p53蛋白對A549細胞內生性微型核醣核酸-18a表現之影響-44
圖七(a) 人類女性肝癌檢體 (HBV陽性) 之p53蛋白、p53蛋白之RNA
及成熟微型核醣核酸-18a之表現量 (組別: 成熟微型核醣核酸-18a於腫瘤部分上升大於三倍)-45
圖七(b) 人類女性肝癌檢體 (HBV陽性) 之p53蛋白、p53蛋白之RNA
及成熟微型核醣核酸-18a之表現量 (組別: 成熟微型核醣核酸-18a於腫瘤部分上升小於三倍)-46
圖八 以定序方法偵測臨床檢體中之p53蛋白突變-47
圖九 (a) 以西方墨點法偵測具點突變之p53蛋白之穩定性-48
圖九 (b) 以西方墨點法偵測具點突變之p53蛋白之穩定性(量化後作圖)-49
圖十 自臨床檢體中辨識到之突變型p53亦可促進成熟微型核醣核酸-18a之表現-50
圖十一(a) 人類女性肝癌檢體 (HBV陽性) 之p53蛋白及成熟微型核醣核酸-18a之表現量-51
圖十一(b) 人類女性肝癌檢體 (HCV陽性) 之p53蛋白及成熟微型核醣核酸-18a之表現量-53
圖十二 成熟微型核醣核酸-18a於HBV陽性或HCV陽性之女性肝癌中之
上升情形-55
圖十三 臨床檢體中p53蛋白累積與成熟微型核醣核酸-18a表現上升之相關性具病毒特異性-56
表一 可影響processing之因子整理-57
表二 HBV陽性檢體p53蛋白定序結果-58

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