肝細胞癌(Hepatocellular carcinoma, HCC)是全球常見的惡性腫瘤之一，並且也是全球第三名造成死亡的癌症。造成肝細胞癌的風險因子包括過度飲用酒精、黃麴毒素以及肝臟相關病毒的感染為主。雖然現今的醫學已有許多治療肝細胞癌的方法，可是對於肝癌的高復發率卻沒有有效的方法進行預防。有許多研究團隊漸漸開始重視這個問題，因此我們實驗室也希望能透過臨床檢體的分析找尋有潛能的預後因子，我們利用了肝細胞癌進行全基因組微陣列分析，成功篩選出在肝癌腫瘤組織中過表達的基因，其中我們發現了Never in mitosis A-related kinase 2 (Nek2)，一個與細胞週期相關的基因，Nek2的功能是參與在中心體分離和有絲分裂的細胞中雙極晶體管形成的一個重要激酶。先前實驗室成員透過RT-PCR分析了97位 HCC病人檢體，結果發現在腫瘤組織中的Nek2 mRNA表達比非腫瘤組織中高出許多，這表明Nek2與肝癌發展過程有關。另外利用多變相分析了肝細胞癌患者術後的復發時間與Nek2表現量的關係，發現在Nek2表現量高的族群中相對復發率也高。為了探討Nek2在肝癌進展中的角色，利用已建立的Nek2 overexpression及knockdown細胞株進行實驗。在colony assay及BrdU incorporation assay中，我們發現Nek2會幫助細胞的生長週期增快。此外在migration assay發現Nek2會增加細胞的爬行能力，並且細胞中E-cadherin的表現會隨著Nek2的下降而增加。而在invasion assay中發現Nek2對於細胞的侵犯能力有增強的影響，代表Nek2的表現促使細胞傾向表現癌細胞轉移的特性。在先前研究也發現， Nek2 knockdown促使細胞在G1 / S期的細胞週期停滯，而我們發現當Nek2 knockdown時會促使抑制 Akt的磷酸化以及p27的大量表現，Akt是一個已知磷酸化後可將p27蛋白降解，因此我們認為Nek2是透過調節Akt磷酸化和p27的降解促使細胞週期的停滯。總結上述所發現的，我們認為Nek2 確實在肝細胞癌中扮演重要的角色，並且調控細胞表現出惡性癌細胞增生及轉移的特性，這可能是Nek2導致肝癌患者術後預後不佳的因素之一。

Summary
In this study, we found that the levels of Nek2 expression could up-regulate the cell proliferation, migration and invasion. The down-regulation of Nek2 induces cell cycle arrest in HCC cell, though increase Akt phosphorylation and p27 degradation.
Key words: Hepatocellular carcinoma, cell cycle, migration, proliferation.

Introduction
Hepatocellular carcinoma (HCC) is one of the most common malignancies and represents the third-leading cause of cancer-related death worldwide. The risk factors for HCC include alcohol, toxins, and HBV and HCV infections. We recently performed whole-genome cDNA microarray analysis in HCC and identified some genes that were over-expressed in tumorous tissues. Among them, Never in mitosis A-related kinase 2 (Nek2), an essential factor for centrosome separation and bipolar formation in mitotic cells, was hypothesized to play important roles in HCC progression. By the analysis of real-time RT-PCR in 97 HCC cases, it was found that the Nek2 mRNA expression in tumorous tissues was higher than the non-tumorous tissues, suggesting that Nek2 is associated with HCC carcinogenic processes. The aim of this study is to delineate the molecular mechanism of Nek2 in regulation of HCC progression.
Materials and Methods
In this study, we set up the Nek2 overexpression and knockdown stable cell line to understand the cell phenotype. The experimental methods include colony assay, BrdU incorporation assay, transwell migration assay, wound healing assay, and invadopodia assay. We also use western blot to determine the expression of cell cycle related proteins.

Results and Discusson
In the colony formation assays, the numbers of HuH7 colonies were positively affected by the Nek2 expression. Furthermore, the cell proliferation rates, analyzed using the BrdU incorporation assays, found that cells that were stably transfected with Nek2 expression showed increased cell proliferation. We also found that the migration rates of Nek2 expression cells were more than control cells by analyzed using the transwell assay and wound healing assay.
In the other hand, we found that Nek2 knockdown in HuH7 cells promoted G1/S cell cycle arrest, indicating that the Nek2 is involved in cell cycle G1/S progression. We also found that cyclin dependent kinase inhibitor p27, important regulator in G1 to S progression, was up-regulated in Nek2 knockdown cells. In addition, Akt phosphorylation, known to enhance p27 degradation, was reduced in these cells. Based on these findings, we suggest that Nek2 regulates Akt phosphorylation and p27 degradation to enhance cell cycle progression.
Conclusion
In this study, we analyzed the cell phenotype in Nek2 overexpression and knockdown cells. These data suggested that Nek2 plays an important role of hepatoma progression.

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