Objectives: Liver cancer is the sixth most common malignant tumor in the world. Hepatocellular carcinoma (HCC) accounts for 85%-90% of all patients with liver cancer. It possesses the characteristics of insidious onset, rapid progression, early recurrence, easy drug resistance, and poor prognosis. NIMA related kinase 2 (NEK2) is a cell cycle regulating kinases, which regulates cell cycle in mitosis. Cellular senescence is a complex heterogeneous process, and is a stable form of cell cycle arrest that limits the proliferative potential of cells. This study aims to investigate the relationship between the expression level of NEK2 and the senescence in hepatoma cells, and to explore the effect of NEK2 expression on hepatoma cell senescence and the underlying molecular mechanism.
Methods: A total of 581 senescence-relevant genes were obtained from the GenAge website. The gene expression data of tumor tissues of 370 HCC patients were downloaded from the Cancer Genome Atlas database. The co-expression of NEK2 and aging-related genes was analyzed by R-package. KEGG was used to analyze the significant gene enrichment pathway of differentially expressed genes in NEK2 overexpression HEK293. The stable transfected cell lines with overexpression and knockdown of NEK2 were constructed in hepatoma cell line SMMC-7721 and HepG2, and senescence-associated β-galactosidase (SA-β-gal) staining was used to detect senescence, the cell proliferation was detected by CCK-8 method and clone formation experiment, the cell cycle was analyzed by flow cytometry, and the expression of proteins related to p53/p21, p16/Rb, and phosphatase and tensin homolog deleted on chromosome ten (PTEN)/Akt signal transduction pathway was detected by Western blotting.
Results: There were 320 senescence related genes co-expressed with NEK2. KEGG analysis showed that the senescence signaling pathway was significantly enriched in HEK293 cells with overexpression of NEK2.Compared with SMMC-7721 or HepG2 without knockdown of NEK2, the senescent cells of SMMC-7721 and HepG2 with knockdown of NEK2 were increased, cell proliferation and clone formation were decreased significantly, the percentage of cells in G0/G1 phase was increased, the expression levels of phospho-Akt (p-Akt) and phospho-Rb (p-Rb) protein were decreased significantly, and the expression level of p16 protein was increased significantly (all P<0.05). Compared with SMMC-7721 or HepG2 transfected with blank plasmid, the senescent cells of SMMC-7721 and HepG2 overexpressing NEK2 were decreased, the cell proliferation and clone formation were increased significantly, the percentage of cells in G0/G1 phase were decreased, the expression levels of p-Akt and p-Rb protein were increased significantly, and the expression level of p16 protein was decreased significantly (all P<0.05).
Conclusions: NEK2 may mediate the anti-aging effect of hepatoma cells through p16/Rb and PTEN/Akt signal transduction pathways, which provides a new theoretical basis for NEK2 to promote the progress of liver cancer and a new idea for the targeting treatment for liver cancer.
目的: 肝癌是全球第6大常见的恶性肿瘤，肝细胞癌(hepatocellular carcinoma，HCC)占所有肝癌患者的85%~90%。HCC具有起病隐匿、进展快、复发早、易耐药和预后差的临床特点。中心体相关激酶2(NIMA related kinase 2，NEK2)是细胞周期调控蛋白激酶，在有丝分裂中调节细胞周期。细胞衰老是一个复杂的异质过程，是一种稳定的细胞周期停滞形式，可限制细胞的增殖潜能。本研究旨在分析NEK2表达水平与肝癌细胞衰老的关系，探究NEK2对肝癌细胞衰老的影响及其分子机制。方法: 从GenAge获得581个衰老相关基因，从癌症基因组图谱数据库下载370例HCC患者肿瘤组织的基因表达数据，利用R包分析NEK2与衰老相关基因的共表达情况。采用KEGG分析NEK2过表达的HEK293细胞差异基因的富集通路。在肝癌细胞SMMC-7721及HepG2中构建NEK2过表达及敲低的稳定转染细胞株，采用衰老相关的β-半乳糖苷酶染色检测衰老细胞，CCK-8法和克隆形成实验检测细胞增殖，流式细胞仪分析细胞周期，蛋白质印迹法检测p53/p21、p16/Rb和第10号染色体上缺失与张力蛋白同源的磷酸酯酶(phosphatase and tensin homolog deleted on chromosome ten，PTEN)/Akt信号转导通路相关蛋白质的表达情况。结果: 与NEK2共表达的衰老相关基因有320个。KEGG分析显示细胞衰老信号通路在NEK2过表达的HEK293细胞中存在显著富集。与未敲低NEK2的SMMC-7721或HepG2相比，敲低NEK2的SMMC-7721和HepG2衰老细胞增加，细胞增殖和克隆形成明显减少，G0/G1期细胞的百分比增加，磷酸化的Akt(phospho-Akt，p-Akt)和磷酸化的Rb(phospho-Rb，p-Rb)蛋白质表达水平明显降低，p16蛋白质表达水平明显升高(均P<0.05)；与转染空白质粒的SMMC-7721或HepG2相比，过表达NEK2的SMMC-7721和HepG2衰老细胞减少，细胞增殖和克隆形成明显增加，G0/G1期细胞的百分比减少，p-Akt和p-Rb蛋白质表达水平明显升高，p16蛋白质表达水平明显降低(均P<0.05)。结论: NEK2可能通过p16/Rb及PTEN/Akt信号转导通路介导肝癌细胞的抗衰老效应，为NEK2促进肝癌进展提供了新的实验依据，也为肝癌的靶向治疗提供了新思路。.
Keywords: NIMA related kinase 2; cell cycle; cell proliferation; cellular senescence; liver cancer.