NEK2 promotes the progression of liver cancer by resisting the cellular senescence

Abstract

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.

Keywords: NIMA related kinase 2; cell cycle; cell proliferation; cellular senescence; liver cancer.

图1

TCGA数据库中肝癌患者NEK2与衰老基因共表达情况的分析 Figure 1 Analysis of the co-expression of NEK2 and aging genes in patients with HCC in TCGA database NEK2: NIMA related kinase 2; HCC: hepatocellular carcinoma; TCGA: the Cancer Genome Atlas.

图2

差异表达基因富集到细胞周期和细胞衰老信号通路 Figure 2 Differentially expressed genes enriches in signaling pathway of cell cycle and cellular senescence

图3

敲低NEK2促进肝癌细胞SMMC-7721(A)和HepG2(B)的衰老 Figure 3 Knockdown of NEK2 promotes cell senescence in hepatoma cells SMMC-7721 (A) and HepG2 (B) NEK2: NIMA related kinase 2. **P<0.01.

图4

过表达NEK2抑制肝癌细胞SMMC-7721(A)和HepG2(B)的衰老 Figure 4 Overexpression of NEK2 inhibits cell senescence in hepatoma cells SMMC-7721 (A) and HepG2 (B) NEK2: NIMA related kinase 2. *P<0.05, **P<0.01.

图5

敲低NEK2抑制肝癌细胞SMMC-7721(A)和HepG2(B)的增殖 Figure 5 Knockdown of NEK2 inhibits cell proliferation in hepatoma cells SMMC-7721 (A) and HepG2 (B) NEK2: NIMA related kinase 2. **P<0.01.

图6

过表达NEK2促进肝癌细胞SMMC-7721(A)和HepG2(B)的增殖 Figure 6 Overexpression of NEK2 promotes cell proliferation in hepatoma cells SMMC-7721 (A) and HepG2 (B) NEK2: NIMA related kinase 2. *P<0.05, **P<0.01.

图7

敲低NEK2抑制肝癌细胞SMMC-7721(A)和HepG2(B)的克隆形成 Figure 7 Knockdown of NEK2 inhibits clonogenesis in hepatoma cells SMMC-7721 (A) and HepG2 (B) NEK2: NIMA related kinase 2. **P<0.01.

图8

过表达NEK2促进肝癌细胞SMMC-7721(A)和HepG2(B)的克隆形成 Figure 8 Overexpression of NEK2 promotes clonogenesis in hepatoma cells SMMC-7721 (A) and HepG2 (B) NEK2: NIMA related kinase 2. **P<0.01.

图9

敲低NEK2抑制肝癌细胞SMMC-7721(A)和HepG2(B)阻滞细胞周期进程 Figure 9 Knockdown of NEK2 inhibits cell cycle in hepatoma cells SMMC-7721 (A) and HepG2 (B) NEK2: NIMA related kinase 2. *P<0.05, **P<0.01.

图10

过表达NEK2促进肝癌细胞SMMC-7721(A)和HepG2(B)细胞周期进程 Figure 10 Overexpression of NEK2 promotes cell cycle in hepatoma cells SMMC-7721 (A) and HepG2(B) NEK2: NIMA related kinase 2. *P<0.05.

图11

NEK2激活肝癌细胞p16/Rb及PTEN/Akt信号通路 Figure 11 NEK2 activates p16/Rb and PTEN/Akt signaling pathways in hepatoma cells A: Electropherogram of Western blotting showing the protein expression of NEK2, p-Akt, p-Rb, p16, p53, and p21; B: Relative expression of NEK2, p-Akt, p-Rb, p16, p53, and p21 normalised by GAPDH was determined by densitometric analysis. NEK2: NIMA related kinase 2; PTEN: phosphatase and tensin homolog deleted on chromosome ten; p-Akt: phospho-Akt; p-Rb: phospho-Rb. *P<0.05 vs the SMMC-7721 Ctrl2 group; †P<0.05 vs the HepG2 Ctrl2 group; ‡P<0.05 vs the SMMC-7721 EV group; §P<0.05 vs the HepG2 EV group.