腎細胞癌是最致命的泌尿性生殖系統癌症，對於化學、放射與激素療法皆有抗性的產生。超過四分之一的患者在確診的同時已經有轉移的情形發生。一旦發生轉移，患者的五年存活率小於百分之十。在先前的實驗結果證明，膜聯蛋白A2 的表現隨著急性腎損傷發生到恢復過程中有所變化，顯示在恢復過程中膜聯蛋白A2 可能參與調節腎小管細胞的增殖和再生。因此，我們推測膜聯蛋白A2 失調可能導致腎細胞癌的產生。膜聯蛋白A2 是一種鈣依賴的磷脂結合蛋白，可以表現在不同型態的細胞。膜聯蛋白A2 表現量在許多腫瘤組織內有增加的情形，可能具有調節癌細胞功能的相關能力，包括血管生成，增殖，凋亡，細胞遷移，侵襲和黏附等。在腎細胞癌中膜聯蛋白A2 的表達與腫瘤分化程度和臨床療效有所關聯。然而，大部分的結果主要來自於在組織病理診斷上的分析，對於膜聯蛋白A2 如何調控在腎細胞癌行為的機轉仍不甚清楚。在此研究中，我們使用腎臟癌組織晶片的免疫組織化學染色和即時聚合酶鏈鎖反應來分析膜聯蛋白A2的表現量。膜聯蛋白A2 的表現在腎細胞癌臨床檢體上有明顯增加的情形，且高表現可以預測腎細胞癌不良預後的情形。因此，我們利用小髮夾型核醣核酸 (shRNA) 慢病毒系統來降低腎細胞癌細胞株的膜聯蛋白A2 基因表現，探討膜聯蛋白A2 在調控腎細胞癌腫瘤惡性特徵所扮演的角色和了解其相關的致病機轉。我們發現膜聯蛋白A2 的表現量降低時會抑制細胞移動和侵襲的能力，但對於細胞增殖的影響較小。降低膜聯蛋白A2的表達，會導致細胞極性的改變和破壞肌動蛋白絲的形成，以及降低的CXCR4表達。此外，膜聯蛋白A2 通過抑制Rho 相關的蛋白酶 (ROCK) 訊息傳遞提高細胞的運動能力，並調節肌動蛋白細胞骨架的動態重組。我們進一步研究膜聯蛋白 A2 在腎細胞癌化學療法抗性所扮演之角色，發現降低膜聯蛋白 A2 的表現，可能藉由抑制細胞自噬作用的調控，使腎癌細胞對於細胞分裂抑制劑 (vincristine) 產生抗藥性。總而言之，我們的研究結果表明腎細胞癌中膜聯蛋白A2 的表現量可以用來作為不良預後的標記，且可以調節細胞移動和化療抗藥性。膜聯蛋白A2 可能作為潛在的腎細胞癌治療標靶。

Renal cell carcinoma (RCC) is the most lethal of urologic malignancies and intrinsically resistant to chemotherapy, radiotherapy and hormone therapy. More than one fourth of
patients have metastasis at presentation. Once metastatic RCC occurs, the 5-year survival rate in metastatic patients is less than 10%. In our pervious study, we had identified Annexin A2 (ANXA2) has been modulated in a temporal pattern concomitant with the initiation and recovery of AKI, and concludes that ANXA2 may play a role in the regulation of renal tubular cell proliferation and regeneration in the recovery process. Therefore, we inferred that the dysregulation of ANXA2 might contribute to the progression of RCC. ANXA2 is a calciumdependent, phospholipid-binding protein found on various cell types. It is overexpression in various tumor types and plays multiple roles in regulating cellular functions, including angiogenesis, proliferation, apoptosis, cell migration, invasion and adhesion. ANXA2 expression was correlated with tumor differentiation and clinical outcome in RCC. However,most of studies have been performed only in histological analysis, and little is known about the subsequent effects of ANXA2 manipulation on RCC behaviors. In this study, we used commercial RCC tissue microarray arrays and quantitative polymerase chain reaction (qPCR) array were used to examine ANXA2 expression level by immunohistochemistry and real-time
polymerase chain reaction analysis. ANXA2 expression levels were increasing in RCC clinical specimens, and high expression might predict poor clinical outcome. Therefore, we used short hairpin RNA (shRNA) – based lentiviral system to evaluate the impact of ANXA2
in modulating the malignant phenotypes in RCC cell lines and further delineate the possible mechanisms in these regulations. Our data indicated that silencing ANXA2 expression presented higher suppression abilities of cell motility and invasion in RCC cells, but had less
effect on the ability of cell proliferation. Silencing ANXA2 expression caused the alterations of cell polarity and disrupted the formation of actin filaments and reduced CXCR4 expression. In addition, ANXA2 promotes RCC cell motility through inhibition of Rho-associated protein kinase (ROCK) signaling and regulates the dynamic reorganization of the actin cytoskeleton.
We further examined the role of ANXA2 for chemotherapy resistance in RCC, and found that
the silencing of ANXA2 increased resistance to cell division inhibitor by inhibiting autophagy
regulation. Overall, our findings indicated that evaluated ANXA2 expression represented a
poor prognosis marker in RCC and the regulatory function of ANXA2 in cell motility and chemoresistance. ANXA2 may be a potential therapeutic target for the treatment of RCC.

目錄 ....................................................................................................................................... I-II
中文摘要 ................................................................................................................................... 1
Abstract ................................................................................................................................. 2-3
1. 背景 ................................................................................................................................ 4-9
1.1 腎細胞癌 .................................................................................................................... 4
1.2 腎細胞癌和急性腎損傷（Acute Kidney Injury）的相關性 ................................... 6
1.3 膜聯蛋白A2 .............................................................................................................. 7
1.4 膜聯蛋白A2 和腎細胞癌的關係 ............................................................................. 9
2. 實驗材料與方法 ......................................................................................................... 10-14
2.1 組織晶片和免疫組織化學染色分析 ...................................................................... 10
2.2 細胞株和培養 .......................................................................................................... 10
2.3 流式細胞技術 .......................................................................................................... 11
2.4 細胞增殖分析 .......................................................................................................... 11
2.5 反轉錄聚合連鎖反應 .............................................................................................. 11
2.6 西方墨點法分析 ...................................................................................................... 12
2.7 傷口癒合分析 .......................................................................................................... 12
2.8 細胞侵襲分析 .......................................................................................................... 12
II
2.9 G-/F- actin (球狀/絲狀肌動蛋白) 比例分析 ......................................................... 13
2.10 抑制腎癌細胞Rho 家族的訊息傳遞 ..................................................................... 13
2.11 腎癌細胞藥物處理 .................................................................................................. 14
2.12 統計分析 .................................................................................................................. 14
3. 結果 ............................................................................................................................. 15-18
3.1 膜聯蛋白A2 在腎細胞癌的表現量增加並影響腎細胞癌的預後 ....................... 15
3.2 建立膜聯蛋白A2 靜默的腎細胞癌細胞株 ........................................................... 16
3.3 膜聯蛋白A2 基因表現量下調會抑制腎細胞癌的細胞移動性 ........................... 16
3.4 靜默膜聯蛋白A2 的表現改變腎細胞癌細胞極性和干擾絲狀肌動蛋白的形成 ...
................................................................................................................................. 16
3.5 膜聯蛋白A2 經由抑制Rho 訊息傳遞來促進腎細胞癌的的細胞運動性 .......... 17
3.6 膜聯蛋白A2 基因下調會抑制CXCR4 的表現 .................................................... 17
3.7 膜聯蛋白 A2 的表現影響細胞分裂抑制劑對於腎細胞癌之療效 ...................... 17
3.8 靜默膜聯蛋白 A2 的表現對於腎細胞癌化學和標靶藥物療法療效的影響 ...... 18
4. 討論 ............................................................................................................................. 19-22
5. 結論與展望 ...................................................................................................................... 23
6. 參考文獻 ..................................................................................................................... 24-27
7. 圖表 ............................................................................................................................. 28-50
8. 附錄 ............................................................................................................................. 51-56

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