Regulator of chromosome condensation 1 promotes hepatocellular carcinoma proliferation via cell-division-cycle-associated-8 dependent phosphoinositide 3-kinase/protein kinase B signaling

Abstract

Background: Hepatocellular carcinoma (HCC) ranks among the most prevalent and deadly malignancies, characterized by a high recurrence rate. Regulator of chromosome condensation 1 (RCC1) serves as a principal guanine nucleotide exchange factor for ras-related nuclear protein guanosine triphosphatase (GTPase) and is implicated in various cancers. However, the role of RCC1 in HCC remains unexplored.

Aim: To elucidate the functional significance and molecular mechanisms of RCC1 in HCC.

Methods: Bioinformatics were to examine the expression levels of RCC1 in HCC and to assess its impact on the prognosis of this malignancy. The cell counting kit-8 assay and flow cytometry were utilized to evaluate the cell viability and cell cycle of HCC cells. Furthermore, quantitative reverse transcription and immunoblotting were to investigate the influence of RCC1 on cyclin associated proteins.

Results: Bioinformatics analysis revealed that RCC1 was highly expressed in HCC and correlated with poor prognosis in HCC patients. Functional studies showed that RCC1 overexpression promoted the malignant phenotype of HCC cells, especially the proliferation of HCC cells, whereas RCC1 knockdown had the opposite effect. Mechanistically, we identified cell division cycle-associated (CDCA) 8 as a downstream target of RCC1 in HCC. RCC1 overexpression markedly increased CDCA8 levels, consequently enhancing cell proliferation and survival in HCC cells. Additionally, we discovered that RCC1 contributed to the development and progression of HCC by activating the phosphoinositide 3-kinase/protein kinase B/cyclin-dependent kinase inhibitor 1a pathway through CDCA8.

Conclusion: Our study provides profound insights into the pivotal role of RCC1 in HCC and its potential as a therapeutic target.

Keywords: Cell cycle; Cell division cycle-associated 8; Hepatocellular carcinoma; Phosphoinositide 3-kinase/protein kinase B pathway; Proliferation; Regulator of chromosome condensation 1.

Figure 1

Bioinformatics analysis shows regulator of chromosome condensation 1 expression and its association with poor prognosis in hepatocellular carcinoma patients. ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. A: Regulator of chromosome condensation 1 (RCC1) expression levels in The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium databases; B: Prognostic analysis of high vs low RCC1 expression in TCGA and International Cancer Genome Consortium databases; C: Pathologic grade analysis of high vs low RCC1 expression in TCGA database; D: Volcano plot of differentially expressed genes between high and low RCC1 expression in TCGA database; E: Heatmap displaying top 50 upregulated genes and downregulated genes with greatest differential changes; F: Functional enrichment analysis: Gene Ontology term enrichment results of differentially upregulated genes; G: Functional enrichment analysis: Kyoto Encyclopedia of Genes and Genomes pathway enrichment of differentially upregulated genes; H: Spearman correlation analysis of gene expression and pathway scores. TCGA: The Cancer Genome Atlas; ICGC: International Cancer Genome Consortium; RCC1: Regulator of chromosome condensation 1; GO: Gene Ontology.

Figure 2

Regulator of chromosome condensation 1 overexpression promotes the cell growth and motility of hepatocellular carcinoma cells. ^cP < 0.001, ^dP < 0.0001. A: Immunoblotting analysis of regulator of chromosome condensation 1 (RCC1) in vector and RCC1-overexpressing Huh-7 cells, with actin as the loading control; B: Quantification of RCC1 expression normalized to actin according to Figure 2A; C and D: Cell counting kit-8 assay and 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay detected the proliferation in vector and RCC1-overexpressing Huh-7 cells; E: Statistical results of 5-ethynyl-2’-deoxyuridine-positive cell percentage from Figure 2D; F and G: Cell cycle analysis and the statistical results between Huh-7-vector and Huh-7-RCC1 cells; H: Wound healing assay detected the migration in vector and RCC1-overexpressing Huh-7 cells; I: Statistical results of wound closure percentage from Figure 2H; J: Transwell invasion assay detected the invasion in Huh-7-vector and Huh-7-RCC1; K: Statistical results of cell numbers from Figure 2J. RCC1: Regulator of chromosome condensation 1.

Figure 3

Knockdown of regulator of chromosome condensation 1 inhibits the proliferation and motility of hepatocellular carcinoma cells. ^cP < 0.001, ^dP < 0.0001. A: Immunoblotting analysis of regulator of chromosome condensation 1 (RCC1) in small interfering RNA negative control (siNC) and RCC1-knockdown Huh-7 cells. Actin was used as the loading control; B: Quantification of RCC1 expression normalized to actin according to Figure 3A; C and D: Cell counting kit-8 assay and 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay detected proliferation in siNC and RCC1-knockdown Huh-7 cells; E: Statistical results of 5-ethynyl-2’-deoxyuridine-positive cell percentage from Figure 3D; F and G: Cell cycle analysis and the statistical results between Huh-7-siNC and Huh-7-siRCC1; H: Wound healing assay detected the migration in siNC and RCC1-knockdown Huh-7 cells; I: Statistical results of wound closure percentage from Figure 3H; J: Transwell invasion assay detected the invasion in Huh-7-siNC and Huh-7-siRCC1; K: Statistical results of cell numbers from Figure 3J. siNC: Small interfering RNA negative control; RCC1: Regulator of chromosome condensation 1.

Figure 4

Regulator of chromosome condensation 1 modulates the expression of cell cycle-associated genes in hepatocellular carcinoma. ^aP < 0.05, ^dP < 0.0001. A: Heatmap displaying the genes correlated with regulator of chromosome condensation 1 (RCC1) in hepatocellular carcinoma samples from The Cancer Genome Atlas database; B: Enrichment analysis of genes with correlation coefficients exceeding 05; C-E: The mRNA and protein expression analysis of cell division cycle-associated (CDCA) 8, minichromosome maintenance complex component, cyclin B1 and CDCA5 in vector and RCC1-overexpressing Huh-7 cells; F-H: Quantitative reverse transcription polymerase chain reaction and immunoblotting analysis of CDCA8, minichromosome maintenance complex component 4, cyclin B1 and CDCA5 in small interfering RNA negative control and RCC1-knockdown Huh-7 cells. RCC1: Regulator of chromosome condensation 1; CDCA: Cell division cycle-associated; MCM4: Minichromosome maintenance complex component 4; CCNB1: Cyclin B1; siNC: Small interfering RNA negative control.

Figure 5

Silencing cell division cycle-associated 8 attenuates the proliferative enhancement induced by regulator of chromosome condensation 1 overexpression in hepatocellular carcinoma cells. ^dP < 0.0001. A: Spearman correlation analysis of regulator of chromosome condensation 1 with cell division cycle-associated (CDCA) 8, minichromosome maintenance complex component 4, cyclin B1 and CDCA5 expression; B: Immunoblotting analysis of the interaction between regulator of chromosome condensation 1 and CDCA8 in Huh-7 cell line; C and D: Cell counting kit-8 assay and 5-ethynyl-2’-deoxyuridine incorporation assay detected proliferation in Huh-7 cells under different treatments; E: Statistical results of ethynyl-2’-deoxyuridine-positive cells percentage from Figure 5D; F and G: Cell cycle analysis and the statistical results in different treatment Huh-7 cells. RCC1: Regulator of chromosome condensation 1; CDCA: Cell division cycle-associated; IP: Immunoprecipitation; siNC: Small interfering RNA negative control; IgG: Immunoglobulin G.

Figure 6

Regulator of chromosome condensation 1 regulates phosphoinositide 3-kinase/protein kinase B/cyclin-dependent kinase inhibitor 1a pathway through cell division cycle-associated 8 in hepatocellular carcinoma. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP< 0.0001. A: Immunoblotting analysis of phosphatidylinositol - 4,5 - bisphosphate 3 - kinase catalytic subunit alpha (PIK3CA), protein kinase B (Akt), p-Akt and cyclin-dependent kinase inhibitor 1a (p21) in vector and regulator of chromosome condensation 1-overexpressing Huh-7 cells. Actin was used as the loading control; B: Quantification of PIK3CA, Akt, p-Akt and p21 expression normalized to actin; C: Immunoblotting analysis of PIK3CA, Akt, p-Akt and p21 in small interfering RNA negative control and regulator of chromosome condensation 1-knockdown Huh-7 cells. Actin was used as the loading control; D: Quantification of PIK3CA, Akt, p-Akt and p21 expression normalized to actin; E: Immunoblotting analysis of PIK3CA, Akt, p-Akt and p21 in different treatment Huh-7 cells. Actin was used as the loading control; F: The quantification of PIK3CA, Akt, p-Akt and p21 expression normalized to actin. RCC1: Regulator of chromosome condensation 1; siNC: Small interfering RNA negative control; CDCA: Cell division cycle-associated; PIK3CA: Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; Akt: Protein kinase B.