Fig 2: The expression of cell division cycle associated 2 (CDCA2) in hepatocellular carcinoma (HCC) tissues and cells. (A) The mRNA levels of CDCA2 in hepatocellular carcinoma (HCC) and paraneoplastic tissues in patients. (B) Protein levels of CDCA2 in four pairs of HCC and paraneoplastic tissues; quantitative analysis was performed with ImageJ software. (C) Immunohistochemistry (IHC) staining showed that CDCA2 was expressed in the nucleus of HCC cells. (D) Morphological analysis showed a positive average rate of 78.8% for CDCA2 in HCC and 2.3% in paraneoplastic tissues. (E) Protein levels of CDCA2 in Huh7, HepG, PLC, and L02 cells; quantitative analysis was performed with ImageJ software. (F) The mRNA levels of CDCA2 in four cells. ***p < 0.001.

Fig 3: Cell division cycle associated 2 (CDCA2) foundations via p53. (A, B). The mRNA and protein levels of the proliferation- and apoptosis-related genes in Huh7 treated with CDCA2 Si-1/2. (C, D) The proliferation of CDCA2 Si-1/2-treated Huh7 cells and LV-CDCA2-treated L02 cells at different time points detected by Cell Counting Kit-8 (CCK8) assay. (E, F) The mRNA and protein levels of the proliferation- and apoptosis-related genes in L02 treated with LV-CDCA2. *p < 0.05.

Fig 4: Cell division cycle associated 2 (CDCA2) was associated with hepatocellular carcinoma (HCC) prognosis. (A–F) In The Cancer Genome Atlas (TCGA) cases, the impact of TNM stage and CDCA2 on the prognosis of all HCC cases, early HCC cases (TNM stage I/II), and mid- to late-stage HCC cases (TNM stage III/IV) was analyzed by Kaplan-Meier (KM) survival. (G) Forest plot illustrated the hazard ratio (HR) values of CDCA2 and TNM stage by multivariate Cox regression analysis. (H) The nomogram of prognostic 1-, 3-, and 5-year survival rates for HCC was constructed based on CDCA2 expression values [log2 (norm count + 1)] and TNM stage. (I) Receiver operating characteristic (ROC) curve analysis of predictive efficacy of the nomogram model. (J, K) Comparison of mRNA levels and survival analysis of CDCA2 in HCC and paraneoplastic samples originating from Japan in International Cancer Genome Consortium (ICGC) database. ***p < 0.001.

Fig 5: Weighted gene co-expression network analysis (WGCNA) construction and identification of the module containing cell division cycle associated 2 (CDCA2). (A) The WGCNA with group information was constructed through the RNA level of 107 samples in GSE89377, including normal liver (N), chronic hepatitis with low grade and high grade (FL and FH), cirrhosis (CS), dysplastic nodules with low grade and high grade (DL and DH), early-stage HCC (eHCC), and G1, G2, and G3 grade HCC (TG1, TG2, and TG3). (B) Analysis of scale-free fit index and the mean connectivity for various soft-thresholding powers. Testing the scale-free topology when β = 9. (C) Hierarchical clustered expression maps of different genes based on topological overlap, where modules were branches of cluster trees. (D) Heat map of gene topological overlap matrix (TOM) overlap visualization with bright or dark colors representing higher or lower overlap, respectively. (E) In the correlation plot between module genes and clinical groups, each row corresponds to a module, columns represent clinical groups, red represents positive correlation, and green represents negative correlation. The correlation coefficients of the turquoise module were labeled in the figure. (F) Gene enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway was completed by R software.