Cyclin-Dependent Kinase Regulatory Subunit 2 Indicated Poor Prognosis and Facilitated Aggressive Phenotype of Hepatocellular Carcinoma

Abstract

Cyclin-dependent kinase regulatory subunit 2 (CKS2) is a member of the cell cycle-dependent protein kinase subunit family, which is implicated as an oncogene in various malignancies. However, the clinical significance, oncogenic functions, and related mechanisms of CKS2 in hepatocellular carcinoma (HCC) remain largely unclear. In the present study, expression features and prognostic value of CKS2 were evaluated in the bioinformatic databases and HCC tissues. The effects of CKS2 on the malignant phenotypes of HCC cells were explored in vitro. According to the analyses of three bioinformatic databases, mRNA levels of CKS2 were elevated in HCC tissues compared with the normal tissues. Immunohistochemical assays found that high CKS2 expression was closely associated with liver cirrhosis (P = 0.019), poor differentiation (P = 0.02), portal vein invasion (P < 0.001), TNM stage (P = 0.019), tumor metastasis (P = 0.008), and recurrence (P = 0.003). The multivariate regression analyses suggested that CKS2 was an independent prognostic factor for overall survival (HR = 2.088, P = 0.014) and disease-free survival (HR = 2.511, P = 0.002) of HCC patients. Moreover, the bioinformatic analyses indicated that CKS2 might be associated with the malignant phenotypes in HCC progression. In addition, in vitro assays showed that CKS2 expression was higher in HCC cell lines than in normal liver cells. Knockdown of CKS2 remarkably repressed the proliferation, colony formation (P = 0.0003), chemoresistance, migration (P = 0.0047), and invasion (P = 0.0012) of HCC cells. Taken together, overexpression of CKS2 was significantly correlated with poor prognosis of HCC patients and the malignant phenotypes of HCC cells, suggesting that it was a novel prognostic biomarker and potential target of HCC.

Figure 1

Upregulated CKS2 mRNA in HCC tissues. CKS2 mRNA levels in HCC tissues, normal liver tissues, or tissues with chronic liver diseases were extracted from several bioinformatic databases, including TCGA (a), GSE14520 (b), GSE45436 (c), GSE36376 (d), GSE54238 (e), and Oncomine (f). All data were normalized with log2. HCC: hepatocellular carcinoma; CKS2: cyclin-dependent kinase regulatory subunit 2. ^∗∗P < 0.01.

Figure 2

CKS2 expression in HCC tissues and paracancerous tissues by immunohistochemistry. (a) Representative immunohistochemical staining images of paracancerous tissues and HCC tissues in 156 HCC cases at different TNM stages. (b, c) Semiquantitative analysis was conducted to assess the CKS2 protein levels between HCC and paracancerous tissues or among HCC cases at different stages. HCC: hepatocellular carcinoma; para: paracancerous tissues; CKS2: cyclin-dependent kinase regulatory subunit 2; TNM: tumor node metastasis. ^∗∗P < 0.01.

Figure 3

Overall survival and disease-free survival curves for 156 HCC patients. (a) Kaplan-Meier analysis of overall survival (OS) according to high or low CKS2 expression in 156 HCC patients. (b) Kaplan-Meier analysis of disease-free survival (DFS) according to high or low CKS2 expression in 156 HCC patients. (c) Kaplan-Meier curves of OS according to high or low CKS2 expression in the TCGA cohort. (d) Kaplan-Meier curves of DFS according to high or low CKS2 expression in the TCGA cohort. CKS2: cyclin-dependent kinase regulatory subunit 2.

Figure 4

Potential roles of CKS2 in HCC progression. (a) The possible roles of CKS2 in tumors were analyzed by the Cancer Hallmarks Analytics Tool. (b) Then, protein interaction analysis of CKS2 was predicted by the GeneMANIA tool. (c) GO annotations based on the top 116 upregulated and downregulated genes associated with CKS2 expression levels. (d) KEGG pathway analysis based on the top 116 upregulated and downregulated genes that were associated with CKS2 expression levels. (e) Gene set enrichment analysis (GSEA) of TCGA datasets elucidated that CKS2 was implicated in cell cycle and DNA replication pathways. (f) The correlation of CKS2 with proliferative markers CCNB1, PCNA, and Ki-67 was analyzed by the GEPIA tool. CKS2: cyclin-dependent kinase regulatory subunit 2; NES: normalized enrichment score.

Figure 5

CKS2 promoted malignant behaviors of HCC cells. (a) The CKS2 expression of HCC cell lines was detected by western blotting. (b) The intensity of each bar in (a). (c) The CKS2 mRNA expression in HCC cell lines was detected by RT-qPCR. (d) CKS2 expression of MHCC97H cells transfected with KD-CKS2 plasmid was measured by western blotting. (e) The intensity of each bar in (d). (f) CKS2 expression of MHCC97H cells was detected by RT-qPCR. (g) CCK8 assay was conducted to determine the proliferation of MHCC97H cells transfected with NC and KD-CKS2. (h) The representative images of MHCC97H cell-derived colonies. (i) The colony number derived from cells transfected with NC or KD-CKS2. (j) The viability of MHCC97H cells with sorafenib treatment. (k) The viability of MHCC97H cells with regorafenib treatment. (l) The representative images of migration cells in the Transwell assay. (m) The number of migration cells. (n) The representative images of invasive cells in the Transwell assay. (o) The number of invasive cells. CKS2: cyclin-dependent kinase regulatory subunit 2. ^∗P < 0.05; ^∗∗P < 0.01.