NEK2 serves as a prognostic biomarker for hepatocellular carcinoma

Abstract

Never in mitosis gene A (NIMA)-related kinase 2 (NEK2) is a microtubule-associated protein that regulates spindle assembly in human cells and is overexpressed in various malignancies. However, the role of NEK2 in hepatocellular carcinoma (HCC) remains undetermined. We performed RNA-seq of the HCC cell line SMMC-7721 and the normal liver cell line HL-7702 using the Ion Proton System. NEK2 expression was detected using quantitative reverse transcription polymerase chain reaction in two cell lines and 5 matched HCC and adjacent non-tumorous liver tissues. The correlation between survival and NEK2 expression was analyzed in 359 patients with HCC using RNASeqV2 data available from The Cancer Genome Atlas (TCGA) website (https://tcga-data.nci.nih.gov/tcga/). The expression of NEK2, phospho-AKT and MMP-2 was evaluated by immunohistochemistry in 63 cases of HCC and matched adjacent non-tumorous liver tissues. Relationships between protein expression and clinicopathological parameters were assessed, and the correlations between NEK2 with phospho-AKT and MMP-2 expressions were evaluated. A total of 610 differentially expressed genes (DEGs) were revealed in the transcriptome comparison, 297 of which were upregulated and 313 were downregulated in HCC. NEK2, as the most obviously different DEG in cells and tissues from the RNA-seq data, was listed as an HCC candidate biomarker for further verification. NEK2 was overexpressed in HCC cells and tissues (P=0.002, P=0.013) and HCC patients with a high expression of NEK2 had a poor prognosis (P=0.0145). Clinical analysis indicated that the overexpression of NEK2 in HCC was significantly correlated with diolame complete (P<0.001), tumor nodule number (P=0.012) and recurrence (P=0.004). NEK2 expression was positively correlated with the expression of phospho-AKT (r=0.883, P<0.01) and MMP-2 (r=0.781, P<0.01). Overexpression of NEK2 was associated with clinicopathological characteristics and poor patient outcomes, suggesting that NEK2 serves as a prognostic biomarker for HCC. Alteration of NEK2 protein levels may contribute to invasion and metastasis of HCC, which may occur through activation of AKT signaling and promotion of MMP-2 expression.

Figure 1

The common differential genes between cell DEGs and tissue DEGs. (A) Venn diagram of the overlaps between cell DEGs and tissue DEGs. The red circle represents 610 DEGs between HCC cells and normal liver cells. The blue circle represents 1378 DEGs between HCC tissues and adjacent non-tumorous liver tissues. There were 12 common differential genes between the two circles. (B) Relative expression of 12 common differential genes in HCC cell line SMMC-7721, when compared with normal liver cell HL-7702. (C) Relative expression of 12 common differential genes in HCC tissues, when compared with adjacent non-tumorous liver tissues.

Figure 2

Expression of NEK2 in HCC cell lines and HCC tissues. (A) NEK2 mRNA expression in immortalized normal human liver cell line HL-7702 and HCC cell line SMMC-7721 using quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n=6, P=0.002). (B) NEK2 mRNA expression in HCC tissues and matched adjacent non-tumorous liver tissues using qRT-PCR (n=5, P=0.013). (C) Immunohistochemical staining for NEK2 expression in HCC and adjacent non-tumorous liver tissues. Representative images (×200; ×400) of positive staining in HCC tissues and negative staining in adjacent non-tumorous liver tissues. (D) Immunohistochemical staining integrated optical density (IOD) value of NEK2 in HCC and adjacent non-tumorous liver tissues (n=63, P<0.001).

Figure 3

ROC and Kaplan-Meier curves for NEK2 expression in TCGA HCC RNAseq dataset. (A) ROC curve analysis to determine the cut-off point for the high expression of NEK2. The area under curve (AUC) was 0.960 (95% CI, 0.939–0.981), and the cut-off point of NEK2 for high expression was 52.73. (B) Kaplan-Meier curve for HCC patients in high-expression (n=66) and low-expression (n=293) groups segregated by the cut-off point. HCC patients with a high expression of NEK2 had a poor prognosis (log-rank test, P=0.0145).

Figure 4

Immunohistochemical staining of phospho-AKT and MMP-2 expression in HCC and corresponding tumor-adjacent tissues. (A) Immunohistochemical staining for phospho-AKT expression in HCC and adjacent non-tumorous liver tissues. Representative images (×200; ×400) of positive staining in HCC tissues and negative staining in adjacent non-tumorous liver tissues. (B) Immunohistochemical staining integrated optical density (IOD) value of phospho-AKT in HCC and adjacent non-tumorous liver tissues (n=63, P=0.0048). (C) Immunohistochemical staining for MMP-2 expression in HCC and adjacent non-tumorous liver tissues. Representative images (×200; ×400) of positive staining (right) in HCC tissues and negative staining (left) in adjacent non-tumorous liver tissues. (D) Immunohistochemical staining integrated optical density (IOD) value of MMP-2 in HCC and adjacent non-tumorous liver tissues (n=63, P=0.027).

Figure 5

Relationships between NEK2, phospho-AKT, and MMP-2 expression and clinicopathological features in HCC. (A) NEK2, phospho-AKT and MMP-2 expression in the HCC tumor size ≤10 cm group was 1.58-fold (P=0.024), 2.24-fold (P=0.041) and 2.29-fold (P=0.013) higher, respectively, than that in the HCC tumor size >10 cm group. (B) NEK2 and MMP-2 expression in the HCC diolame incomplete group was 1.91-fold (P<0.001) and 2.04-fold (P=0.009) higher, respectively, than that in the HCC diolame complete group, but no obvious change was observed in phospho-AKT expression in the HCC diolame incomplete group (P=0.948). (C) NEK2, p-AKT and MMP-2 expression in the HCC multinodular group was 1.62-fold (P=0.012), 2.40-fold (P=0.046), and 2.04-fold (P=0.024) higher, respectively, than that in the HCC uninodular group. (D) NEK2 and p-AKT expression in the HCC recurrence group was 2.09-fold (P=0.004) and 3.24-fold (P=0.045) higher, respectively, than that in the HCC non-recurrence group, but no obvious change was observed in MMP-2 expression in the HCC non-recurrence group (P=0.992).

Figure 6

NEK2 expression and correlation with phospho-AKT and MMP-2 in HCC tissues. (A) Significant positive correlation between NEK2 expression and phospho-AKT expression (r=0.883; P<0.001). (B) Significant positive correlation between NEK2 expression and MMP-2 expression (r=0.781; P<0.001).