肝癌(Hepatocellular carcinoma, HCC)在世界是最常見的惡性腫瘤之ㄧ， 在台灣，數年來一直位處國人十大死因之榜首。儘早發展出相關的肝癌標記,使能配合現今使用之甲級胎兒球蛋白(Alfa-fetoprotein)而達到早期診斷目的，實為當務之急。在許多的報導中曾證實S-phase protein kinase associated protein 2 (SKP2)蛋白質與其輔助因子CDC28蛋白質激鋂調控次單元1B (CKS1B)表現量的升高，導致它們參與某些週期素依賴型激鋂抑制分子(Cyclin-dependent kinase)的蛋白質降解作用，而導致癌化的發生。因此我們選用一些肝癌細胞株，並收集了66組配對之肝癌病人癌化及正常組織，進行CKS1B於肝癌表現量的評估，並利用專一性抑制功能的小片段干擾RNA (siRNA)達成抑制CKS1B的表現，以此研究是否SK-hep1肝癌細胞株的細胞週期因此發生影響或改變。我們的結果證實了CKS1B mRNA的表現在肝癌細胞株以及組織中有升高的趨勢，但同時CKS1B之蛋白質表現量卻未有同時升高的趨勢，顯示CKS1B蛋白質於肝癌組織中呈現不穩定的狀態，推測可能由於細胞週期內APC/CCHD1 (作用於G0-G1期)所調控之CKS1B降解作用造成此現象。另外，p27蛋白質的低表現量趨勢與肝癌亦有些微相關性。但CKS1B與p27蛋白質於肝癌間亦沒有明顯正或負相關性，顯示應另有其他的路徑調控了此兩種蛋白質於肝癌間的表現。而與p27共同為此蛋白質降解路徑之對象的p21蛋白質，其低表現量顯然與肝癌並不相關，顯示可能有蛋白質後修飾所導致之蛋白質穩定現象調控了p21蛋白質於肝癌的表現。另一方面，CKS1B特異性siRNA對SK-hep1細胞株之細胞週期影響試驗中，在si-CKS1B處理過後之24小時之間，p27及SKP2的蛋白質表現呈上升趨勢，p21呈現初期略為升高但最終下降的趨勢。但在mRNA表現的部份，事實上在si-CKS1B處理之48小時後，p21及p27的mRNA表現皆呈現上升趨勢。我們同時亦偵測了其他相關細胞週期調控因子的mRNA表現情況，結果顯示除了p21, p27和Cyclin D2外，幾乎所有細胞週期調控因子的mRNA表現量在si-CKS1B處理之48小時後皆呈現下降的趨勢。同時亦可看到細胞在此時呈現凋亡的現象，因此我們推測si-CKS1B對CKS1B表現量的抑制，在肝癌細胞株內可達成抑制細胞不正常分化及癌化的效果。依上述結果推測，SCFSKP2-CKS1B蛋白質降解路徑可能並非直接影響並調控肝細胞的癌化。可能存在其他未知的新路徑，伴隨已知的APC/CCHD1 (作用於G0-G1期)和SCFSKP2-CKS1B (作用於G1-S期)蛋白質降解路徑，共同調控肝癌的發生。

Hepatocellular carcinoma (HCC) or hepatoma is the top one cause of death in Taiwan based on the Cause of Death Statistics from the Department of Health, Executive Yuan, Taiwan, for many years. To identify any reliable HCC markers and further applied with the AFP measurement to improve the early diagnosis of HCCs is the most important thing. A high expression level of S-phase protein kinase associated protein 2 (SKP2) protein and its cofactor CDC28 protein kinase regulatory subunit 1B (CKS1B) involved in ubiquitination of some cyclin-dependent kinase (Cdk) inhibitors has been reported in various carcinoma. In this study, we examined the expression of CKS1B in HCC tissues and cell lines, and tested the cell cycle effect caused by specific small interference RNA (siRNA) of CKS1B in SK-hep1 cell line. Up-regulated CKS1B mRNAs in HCC cell lines and tissues were identified in our study, when comparing to the normal liver tissues. But we also found lack of up-regulated CKS1B proteins in our HCC tissues at the same time, indicated that CKS1B proteins might be unstable in HCCs. Down regulation of the Cdk inhibitors p27 was only partially associated with HCCs, and the expressions of CKS1B and p27 were not correlated to each other in HCCs, suggesting other pathway(s) might involve in the regulation(s) of CKS1B and p27 proteins in the HCCs. Down-regulation of the p21 proteins was also found to be not significantly associated with HCCs tissues, this result strongly suggested a post-translational stabilization way might regulate(s) the p21 protein levels in HCCs tissues. On the other hands, in time course experiment, disruption of CKS1B mRNA by si-CKS1B up-regulated the expressions of p27 and SKP2 protein levels and down-regulated the p21 protein level in the SK-hep1 hepatoma cell lines for 24 hrs later. But the mRNA expression level of p21 and p27 were actually both up-regulated for 48 hrs after transfected with si-CKS1B. We also tested the mRNA expression level of many cell cycle regulatory factors for 48 hrs after transfected with si-CKS1B. The results exhibited almost all of the factors (excepted p21, p27 and Cyclin D2) were down-regulated. Furthermore, we saw the apoptosis appearance of SK-hep1 cell after transfected with si-CKS1B for 48 hrs, suggesting the abnormal cell proliferation and tumorigenesis were controlled by siRNA transfection. Taken together, these results suggest that SCFSKP2-CKS1B pathway might not direct involved in ubiquitination of Cdk inhibitors. Another pathway(s), either known or novel, in addition to APC/CCHD1 (G0-G1 phase) and SCFSKP2-CKS1B (G1-S phase) regulation pathways, might regulate the tumorigenesis of HCCs.

Abbreviations………………………………………………………I
Abstract
Chinese…………………………………………………………II
English…………………………………………………………IV
Introduction……………………………………………………… 1
Materials and Methods……………………………………………6
Results………………………………………………………………11
Discussion………………………………………………………… 17
Figures and Tables……………………………………………… 25
References………………………………………………………….54

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