RECK(Reversion-inducing Cysteine-rich protein with Kazal motifs)最早是由日本Noda實驗室於1998年利用表現選殖的方式所找到的一個transformation suppressor gene。目前的研究報導指出RECK可藉由調控MMP-2 和MMP-9進而抑制細胞侵犯性、轉移和血管新生的能力。而RECK通常表現在正常組織及未經轉型的細胞中，在許多經轉型的惡性細胞株中，RECK mRNA的表現有明顯被抑制的情形。也有報導指出ras造成RECK被抑制主要是透過抑制其轉錄，且可能是透過RECK promoter上的SP1 binding site。不過，RECK基因是如何受到抑制的詳細機制，目前尚不清楚。
DNA甲基化的改變是人類腫瘤組織常見的變異，也是造成基因不表現的主要機制之一。腫瘤抑制基因之啟動子區域高度甲基化是腫瘤組織中常見的現象。所以在本論文中，主要在探討Ras造成轉移抑制基因RECK表現的降低和RECK啟動子甲基化之間的相關性。
利用2-12細胞是Lac operator/repressor system 的細胞株，經由IPTG可以誘導ras的表現。首先利用methylation specific PCR的方式探討RECK promoter methylation的情形，經IPTG誘導RECK甲基化有明顯增加的趨勢，進一步利用RT-PCR和western blotting觀察RECK及DNA methyltransferase的表現，結果顯示DNMT3b有明顯增加而RECK表現有下降的情形。所以更進一步利用in vitro (DAPA) and in vivo (ChIP)方法探討RECK promoter和DNMT3b binding affinity的情形是否有所改變，發現在加入IPTG誘導ras表現後，DNMT3b與DNA結合的親合力亦有上升的現象。接著利用DNMT的抑制劑5-AZAC處理，利用MSP、RT-PCR及western blotting的方法觀察加入此抑制劑後的變化，可以看到RECK promoter甲基化的情形下降，且RECK mRNA和蛋白質表現則有回升的情形，而利用DNMT3b siRNA處理 也可以看到相同的結果。
接著探討ras所造成RECK被抑制是透過何種路徑?結果發現當加入U0126 (ERK inhibitor)後，甲基化情形及DNMT3b表現有下降，而RECK表現有上升的現象。在function assay的探討上，利用invasion assay分析5-AZAC及DNMT3b siRNA有抑制2-12細胞侵襲的能力，更進一步在相同5-AZAC條件下利用RECK siRNA處理細胞，發現細胞的侵襲性有明顯增加的情形。
以人類肺癌細胞株(CL1-0及H358)進行實驗時，也可以觀察到相同的結果。我們結論ras所造成RECK的抑制可能是藉由MEK/ERK的路徑，增加DNMT3b和RECK的binding affinity，使RECK promoter甲基化，進而影響RECK基因的表現，使細胞轉移及侵襲的能力增加。

Many tumor suppressor genes are known to be inactivated by epigenetic modifications including DNA methylation and histone deacetylation. RECK is a membrane-anchored glycoprotein that may negatively regulate matrix metalloproteinase (MMP) activity and inhibit tumor metastasis. Our previous study demonstrated that oncogenic ras inhibited RECK expression via Sp1 binding site in the RECK promoter by histone deacetylation mechanism. In this study, we tried to characterize the molecular pathway that mediates the inhibitory effect of ras on RECK. Methylation specific PCR (MSP) indicated that RECK gene promoter is hypermethylated in ras-activated 2-12 cells. We also tested whether ras activation induced the binding of DNA methyltransferases (DNMTs) to Sp1 to repress RECK expression. Our data showed Sp1-associated DNMT3b in cells was increased after ras induction. By using DNA affinity precipitation assay (DAPA) and chromatin immunoprecipitation (ChIP) , we found that induction of oncogenic ras enhanced the binding of DNMT3b to the Sp1 site in the RECK promoter in vitro and in vivo. Additionally, treatment of DNMT inhibitor 5'-azacytidine led to the re-expression of RECK in ras-activated 2-12 cells. The signaling pathway by which ras suppresses RECK was also addressed. Chemical inhibitor of ERK signaling pathway U0126 reversed the methylation of RECK promoter and up-regulated RECK expression in ras-activated 2-12 cells. More importantly, 5'-azacytidine and DNMT3b siRNA may suppress the invasive ability of 2-12 cells. Taken together, our results suggest that oncogenic ras inhibit the metastasis suppressor gene RECK via a DNA methylation mechanism and provide valuable insights for the understanding of the mechanisms by which ras promotes tumor invasion and metastasis

縮寫語------------------------------------------------------------------4
中文摘要---------------------------------------------------------------5
英文摘要---------------------------------------------------------------7
前言---------------------------------------------------------------------8
研究方向---------------------------------------------------------------12
研究材料---------------------------------------------------------------13
實驗方法---------------------------------------------------------------16
實驗結果---------------------------------------------------------------26
討論---------------------------------------------------------------------52
參考文獻---------------------------------------------------------------58
結果圖表---------------------------------------------------------------61
附圖---------------------------------------------------------------------62
致謝辭------------------------------------------------------------------68

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