Cks1在人類非小細胞肺癌組織及細胞株的表現及siRNA對其抑制作用的探討

Study of the expression of Cks1 in human non-small cell lung cancer tissues and cell lines and its inhibition by siRNA

蔡易珊(Yi-Shan Tsai)

指導教授 ： 洪文俊 ； 莊麗月

高雄醫學大學/醫學院/生物化學研究所/碩士(2003年)

摘要

目前已知人類週期素依賴型蛋白激酶次單元（Cks 1）參與細胞週期的進行且也參與p27kip1蛋白分解作用。由於目前尚未清楚Cks 1蛋白在細胞癌化過程中扮演的功能性角色為何，因此我們選用非小細胞肺癌細胞株及組織利用組織免疫染色及具有專一性的siRNA來進行研究。透過免疫組織染色方法在95個肺癌組織發現Cks1有高表現的就佔66 個(比例為69%)，利用反轉錄聚合酶連鎖反應、西方點墨法在細胞株研究發現Cks1也是高度表現。然而在非小細胞肺癌組織及細胞株Cks1與p27kip1的表現是沒有顯著相關的。我們另外選用Cks 1的高表現非小細胞肺癌細胞株（H358）利用轉殖siRNA將Cks1的mRNA分解，以抑制蛋白表現來研究高表現Cks1基因在非小細胞肺癌功能性的角色。我們的結果發現透過專一性抑制功能的siRNA可以抑制Cks1蛋白的表現並進而影響細胞週期的進行，使週期停滯在G2/M時期。其機轉是經由抑制Cdc25C磷酸水解酶的磷酸化及活化，進而抑制cyclinB1/Cdc2的活性，使得細胞週期停滯在G2/M時期。長時間轉殖siRNA會誘導細胞走向凋亡，且不會傷害到正常肺部的細胞。因此我們可以推測高表現Cks1蛋白在非小細胞肺癌的功能性角色可能參與G2/M時期的調控以促進癌細胞增生。因此利用siRNA的技術可以將高表現的Cks1基因專一性的抑制，並誘導細胞走向凋亡，所以siRNA的技術可提供將來發展新的癌症治療方法。

關鍵字

Cks1 ； 非小細胞肺癌

並列摘要

Cdc kinase subunit 1 (Cks1) has been shown to involve in the regulation of cell cycle progression and p27kip1 degradation. However, the functional role of Cks1 in tumorigenesis is still unknown. In this study, we examined the expression of Cks1 in non-small cell lung tumor tissues and cell lines and tested the effect of Cks1 specific small interfering RNA (siRNA) on the proliferation of non-small cell lung cancer cells. Immunohistochemistry analysis showed that strong Cks1 expression was presented in 66 of 95(69%) of human lung tumor tissues. RT-PCR and western blotting analysis also showed that Cks1 was highly expressed in non-small cell lung cancer cell lines. However, no significant correlation between Cks1 and p27kip1 was found in primary non-small cell lung tumor tissues and cell lines. Transfection of Cks1 siRNA induced G2/M arrest in Cks1-overexpressing H358 lung cancer cells, followed by induced of apoptosis in these cells. We also studied the molecular mechanism by which Cks1 siRNA blocked cell cycle progression. Our results suggested that Cks1 siRNA reduced Cdc25C phosphorylation and activation and suppressed cyclinB1/Cdc2 kinase activity to induce G2/M arrest. Long-term treatment of Cks1 siRNA triggered apopotosis in human lung cancer cells. On the contrary, Cks1 siRNA did not affect viability of normal human lung fibroblasts under the same experimental condition. Taken together, these results suggest that Cks1 participates in the steps of lung tumorigenesis and Cks1 siRNA may be used as a therapeutic agent for the treatment of lung cancer.

並列關鍵字

Cks1 ； Non-small cell lung cancer

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