| Summary: | 碩士 === 國立中山大學 === 生物醫學科學研究所 === 93 === 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.
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