Rad9 蛋白質最早是在Schizosaccharomyces prombe 被發現，而進一步在人類身上也同樣發現Rad9的同源蛋白，hRad9，hRad9是checkpoint rad gene的其中一員，是個具有高度保存性的蛋白並且hRad9在細胞中參與了許多不同重要的生理功能。雖然有許多研究文獻報導hRad9蛋白的各種生理功能但是hRad9蛋白在人類細胞癌症中所扮演的真正角色並未明確。細胞老化被認為是細胞保護自己的反應機制之ㄧ並且會出現在較良性的腫瘤中。在之前的相關報導中，在乳癌肺癌中觀察到hRad9蛋白有累積的情形，並且hRad9蛋白被認為是個腫瘤致癌基因；然而，一些研究顯示hRad9蛋白是腫瘤抑制蛋白因為hRad9保持基因的完整度和抑制androgen receptor (AR)的活性。而為了釐清hRad9蛋白在癌症中扮演的角色所以先在MDA-MB-231 breast cancer cell lines 中建立hRad9蛋白大量表現穩定的細胞株（此細胞株已由實驗室凡志學長建立)，下一步我們對於細胞型態、細胞生長速度、細胞爬行能力以及形成聚落的能力進行觀察。結果發現在hRad9大量表現的細胞株中，在細胞型態上可以發現有部分細胞呈現較肥大並有較攤平的外觀並且在細胞生長速率、細胞爬行速度及形成聚落的能力，皆有下降的趨勢。由於細胞的外觀改變並有生長速率的降低的特色和已報導的現象細胞老化( Senescence )相符，故利用其他老化相關特色，例如：Senescence associated β-galactosidase、Stress fibers actin 經由觀察後更進一步發現這類型的細胞有表現老化細胞的特徵出現，藉由這些特徵顯示大量表現hRad9蛋白會使細胞呈現老化的現象；另一方面為了更確定hRad9蛋白的角色定位，我們所取得的invasive ductal carcinoma specimens中有發現大部分檢體呈現hRad9 蛋白低量表現的情形，並且在良性與惡性不同的細胞株中，觀察到較惡性並較具侵犯性的細胞株，hRad9蛋白呈現低表現，這給了我們暗示hRad9在腫瘤惡化程度中可能扮演著重要的角色，因此我們認為hRad9蛋白的下降似乎和細胞較惡化具有一定關係存在，故我們以核酸干擾技術暫時抑制MCF-7 breast cancer cell lines hRad9的表現，從細胞外型中發現在細胞型態上產生改變並且在細胞的爬行能力有上升的表現。並且發現細胞的爬行速率也有上升的情形，進而在西方墨點法技術中，發現Epithelial marker like E-cadherin的減少和Mesenchymal markers like Vimentin的增加，然而此EMT marker的改變是否真的造成在動物model中細胞株轉移能力的增加，還需要進一步的研究探討。綜合以上結果，我們認為hRad9可能透過調控下游因子及EMT相關蛋白進而在乳癌細胞中扮演一個抑癌的角色。而更詳細的機制還必須在未來做更詳盡的探討。

The hRad9 protein is a structural homologue to Schizosaccharomyces pombe rad9 in human beings, which is a member of the Rad family of checkpoints that plays important roles in several fundamental biological processes. Cellular senescence is an anti-proliferative response considered as a tumor suppressive mechanism. Accumulation of hRad9 has been reported in breast, lung and prostate cancers, and suggest that hRad9 may act as an oncogenic protein. However, several studies also support that hRad9 might serve as a tumor suppressor due to its functions in maintaining chromosome integrity and repressing androgen receptor (AR) activity. In this study, We established stably hRad9-overexpressing cells in MDA-MB-231, 231-CR9, and determined their tumorigencity. In comparison with the mock-transfected cells, 231-PC, the morphology of 231-CR9 cell was enlarged and vacuolous. The cell growth rate of 231-CR9 was lower than that of 231-PC cells. In addition, migration abiliyty of 231-CR9 was decreased when compared to that of 231-PC cells. Several features of senescent cells including large size cell , the enrichment stress fibers , and the senescence-associated β-galactosidase (SA-β-gal) was observed in 231-CR9 , and further we knock-down hRad9 expression resulted in the reversal of cellular senescence in 231-CR9, to suggest that hRad9 was associated with cellular senescence in 231-CR9. Knock-down of hRad9 resulted in loss of cell-cell adhesion epithelial markers like E-cadherin , activation of mesenchymal markers, and induction of cell motility. To suggest that reduced hRad9 protein might contribute to metastasis by promoting an epithelial-mesenchymal transition (EMT). Put together, our data indicated that hRad9 protein was involved in regulating senescence and epithelial-mesenchymal transition in cancer cells. We will investigate the mechanisms of hRad9-mediated senescence EMT as well as confirm our hypothesis that hRad9 protein play an important role in promoting senescence and modulating epithelial-mesenchymal transition in vivo in the future.

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