Nek2對肝癌細胞凋亡的影響及其分子機制的研究

廣州醫科大學附屬廣州市第一人民醫院消化疾病中心，廣東 廣州 510180

【Abstract】ObjectiveTo investigate the expression and function of nima-related kinase2 (Nek2) in hepatocellular carcinoma tissues and cell lines, and to study the apoptosis and mechanism of Nek2 in hepatocellular carcinoma.Methods

The expression of Nek2 was detected by Western blotting in 27 hepatocellular carcinoma and 6 human hepatocellular carcinoma cell lines (HepG2, PLC/PRF/5, Hep3B, BEL-7402, SMMC-7721 and QGY-7701 cells), to select the HepG2 cell lines for further experiments. HepG2 cells with Nek2-siRNA inhibitor or the negative control (NC) were transfected, and the biological influence of Nek2-siRNA on HepG2 cells was analyzed in detection of apoptosis by flow cytometry and transfected by Nek2-siRNA for 48 hours, the expressions of apoptosis related factors P53, Caspase3, Bad and Bcl-2 were examined by Western blotting.ResultsNek2 was upregulated in hepatocellular carcinoma tissues and cell lines. Knock down Nek2 lead to promotion of apoptosis. After knock down Nek2 in HepG2 cell, the expressions of apoptosis related factors P53, Caspase3 and Bad were increased, Bcl-2 was reduced.ConclusionInhibiting expression of Nek2 can significantly influence the factors of cell apoptosis signal pathways by increasing P53, Caspase3, Bad, and reducing Bcl-2.

【Keywords】 Nima-related kinase 2; Hepatocellular carcinoma; Apoptosis

近年來，原發性肝細胞癌的發病率有明顯上升的趨勢，全球每年肝細胞癌發病人數為100～150萬人，每年超過60萬人死于肝細胞癌，其在腫瘤死因順位中排名第三位。原發性肝細胞癌患者自診斷之日起中位生存時間少于12個月[1-2]。肝細胞癌的發病機制是一個多因素、多階段、多基因共同積累的過程，其發生、發展涉及多基因調控和多信號通路參與。因此，在腫瘤組織癌變過程中相關基因及基因組的變化研究顯得非常重要。Nek2[never in mitosis A (NIMA)related kinase 2]是一種細胞周期有絲分裂關鍵原件—中心體相關蛋白激酶，Nek2的高表達可導致中心體異常，進而導致腫瘤的發生。目前已經證實在乳腺癌、卵巢癌、宮頸癌、前列腺癌、胃癌、膽管細胞癌和白血病等的人類腫瘤中廣泛存在著Nek2表達升高[3-4]。國內也有報道通過腫瘤組織芯片的免疫組織化學檢測發現，Nek2在消化道腫瘤組織胞漿和胞核中均有高表達，以胞核內高表達為多，所涉及的腫瘤包括食管癌、胃癌、肝癌、小腸癌、結直腸癌及膽管細胞癌。近期有統計[6]指出，高Nek2表達是肝癌患者術后復發的原因之一。

本研究通過檢測Nek2在原發性肝細胞癌組織和肝癌細胞系中的表達和功能，使用小干擾RNA技術，沉默肝癌細胞中的Nek2基因，研究在此前后肝癌細胞凋亡的改變,并初步探索其機制。

1 材料與方法

1.1組織標本及臨床資料新鮮原發性肝細胞癌組織及相應癌旁組織(距離瘤體邊緣>2 cm)共52對，均于液氮速凍后置于-80 ℃冰箱長期保存。其……