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.

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

Core Tip: In this study, we investigated the role of regulator of chromosome condensation 1 (RCC1) in promoting the proliferation of hepatocellular carcinoma (HCC) cells. Initially, we determined that RCC1 is significantly upregulated in HCC and correlates with poor prognosis. Subsequently, we demonstrated that RCC1 facilitates the G₁/S phase transition by regulating cell division cycle-associated 8. Furthermore, our findings indicate that the RCC1/cycle-associated 8 axis promotes HCC proliferation via the phosphoinositide 3-kinase/protein kinase B/cyclin-dependent kinase inhibitor 1a signaling pathway. Collectively, this research elucidates the association between RCC1 and HCC and highlights the role of RCC1 in regulating the G₁/S transition of the cell cycle.

INTRODUCTION

Liver cancer was the sixth most prevalent cancer globally in 2022, with a mortality rate ranking third, following lung cancer and colorectal cancer[1]. Hepatocellular carcinoma (HCC) constitutes over 80% of primary liver cancer cases and represents the most common subtype of liver cancer[2]. Despite the availability of various systemic treatment options for HCC, including atezolizumab in combination with bevacizumab, sorafenib, lenvatinib, and regorafenib[3], the annual incidence and mortality rates remain high, indicating a poor prognosis for the disease[4]. Therefore, further investigation into the mechanisms underlying the onset and progression of HCC is crucial for enhancing the precision of treatment and prognosis of this malignancy.

Regulator of chromosome condensation 1 (RCC1) is the only known guanine nucleotide exchange factor for the ras-related nuclear protein (RAN)[5]. It is implicated in a variety of cellular processes, where it facilitates the activation of Ran by exchanging guanosine diphosphate for guanosine triphosphate (GTP)[6]. This activation is essential for processes including nucleocytoplasmic transport, mitosis, and nuclear membrane formation[7]. Concurrently, RCC1-mediated Ran activation has been demonstrated to accelerate the cell cycle and augment DNA repair mechanisms, thereby alleviating cell senescence induced by DNA damage[8]. Recent research has increasingly highlighted the significant role of RCC1 in tumor biology. A comprehensive pan-cancer analysis has revealed elevated RCC1 expression levels were elevated in multiple tumor tissues, including glioblastoma multiforme, colon adenocarcinoma, kidney renal clear cell carcinoma, liver HCC, and cervical squamous cell carcinoma, compared to normal tissues[9]. Elevated RCC1 expression is positively associated with tumor progression and unfavorable prognosis[10]. In soft-tissue sarcoma, silencing RCC1 expression disrupted the nucleocytoplasmic distribution of S-phase kinase - associated protein 2, thereby inhibiting cell cycle progression, proliferation, and migration[11]. Furthermore, another study that in cervical cancer, suppression of RCC1 Leads to decreased levels of E2F transcription factor 1 protein and impedes the progression of the cell cycle from the G₁ to the S phase, as well as DNA synthesis. Conversely, overexpression of RCC1 eliminated the G₁ checkpoint[12]. These findings indicate that RCC1 is crucial for cell cycle progression and cell proliferation. However, the function of RCC1 in HCC has yet to be explored.

The cell division cycle-associated (CDCA) protein family consists of eight members, CDCA1 through CDCA8, and plays a crucial role in regulating cell division[13]. Notably, CDCA8 is a critical component of the chromosome passenger complex, which also includes survivin, inner centromere protein, and aurora B kinase[14], and is essential for chromosome segregation during mitosis[15]. CDCA8 expression is minimal or weak in most normal tissues[16,17], except in tissues characterized by high cell division activity, such as the testis, lymphoid tissue, and bone marrow. In contrast, CDCA8 is markedly overexpressed in various types of tumor cells[18]. Aberrant expression of CDCA8 has been implicated in the induction of polyploidy, causing failures in cell division, and leading to irregularities in mitotic processes[19]. Empirical research indicates that CDCA8 promotes the proliferation and survival of estrogen-induced breast cancer cells by downregulating the expression of cyclin-dependent kinase inhibitor 1a (p21) and 1b while upregulating cyclin D1 and B-cell chronic lymphocytic leukemia/lymphoma 2[20]. Furthermore, CDCA8 overexpression has been associated with enhanced proliferation in lung[14] and colorectal cancer cells[21] and may contribute to adverse prognostic outcomes in gastric cancer[22] and HCC[23]. Bioinformatics analyses further suggest a significant correlation between CDCA8 expression and the development, onset, and metastasis of HCC[24,25]. However, the underlying mechanism of CDCA8 overactivation in HCC remains to be elucidated.

The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway is critically involved in the pathogenesis of various cancers, mediating key cellular processes such as proliferation, survival, and migration. Dysregulation and aberrant activation of this pathway are strongly associated with tumor initiation and progression, rendering it a promising target for cancer therapeutics[26]. Inhibitors of the PI3K/Akt pathway primarily comprise small molecule inhibitors targeting PI3K, Akt and mechanistic target of rapamycin[27], which effectively impede pathway activation and consequently suppress tumor growth and survival[28]. Certain inhibitors, such as everolimus and temsirolimus, have received approval from the Food and Drug Administration for the treatment of various malignancies[29]. Despite the initial clinical success of early PI3K/Akt inhibitors, their therapeutic potential is constrained by challenges related to pharmacokinetics, tolerance, and efficacy[30]. Therefore, combination therapy strategies that integrate PI3K inhibitors with radiotherapy, chemotherapy, or other targeted therapies are anticipated to yield enhanced clinical outcomes[26]. The regulatory mechanism of the PI3K/Akt pathway is complex, and the treatment of this pathway requires consideration of various mechanisms that lead to drug resistance, such as genetic mutations, feedback loops and microenvironmental factors[31]. Consequently, an in-depth investigation into the regulatory mechanisms of the PI3K/Akt pathway holds substantial clinical significance for the advancement of targeted drug development.

This study aimed to elucidate the functional significance and molecular mechanisms of RCC1 in HCC. Our findings demonstrated that RCC1 overexpression promoted the proliferation and G₁/S phase transition of HCC cells, whereas RCC1 knockdown had the opposite effect. Furthermore, we discovered that RCC1 contributes to the development and progression of HCC by modulating CDCA8 expression and activating the PI3K/Akt/p21 signaling pathway. Collectively, these results highlight the potential of targeting RCC1 as a therapeutic strategy in HCC.

MATERIALS AND METHODS

Bioinformatics analysis

We obtained STAR-counts data and corresponding clinical information for HCC from The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov). The data were subsequently converted to Transcripts Per Million format and subjected to log₂(Transcripts Per Million + 1) normalization. Samples with both RNA sequencing data and clinical information were retained for further analysis. Statistical analyses were performed using R software, version 4.0.3. A P-value of less than 0.05 was considered statistically significant. RCC1 expression levels were categorized into two groups based on the median expression value. The log-rank test was used to compare survival differences between the two groups in the Kaplan-Meier survival analysis. Additionally, both the log-rank test and univariate Cox regression were used to determine the P-value for the Kaplan-Meier curve. To further investigate the role of RCC1 in HCC, we performed differential gene expression analysis, assessed the Gene Ontology (GO) functions of potential genes, and conducted Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The Gene Set Enrichment Analysis algorithm was applied to identify genes and pathways associated with RCC1 expression.

Cell culture

The Huh-7 cell line was obtained from the Japanese Collection of Research Bioresources (JCRB, Osaka, Japan). The cell lines HCC-LM3, AML12 and Hepa1-6 were purchased from the Institute of Cell Biology of Chinese Academy of Sciences (Shanghai, China). The cell lines Huh-7, HCC-LM3 and Hepa1-6 were cultured in RPMI 1640 medium supplemented with 100 mL/L fetal bovine serum (FBS) and 10 mL/L dilution of stock penicillin and streptomycin. The AML12 cell line was cultured in DMEM/F12 medium with 100 mL/L FBS, 10 μg/mL insulin, 5.5 μg/mL transferrin, 5 ng/mL selenium, 50 ng/mL Dexamethasone and 10 mL/L dilution of stock penicillin and streptomycin. All the cell lines were cultured at 37 °C in a humidified 50 mL/L CO₂ incubator.

Transfection

Huh-7 and HCC-LM3 cell lines were transfected with pcDNA3.1-RCC1 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, United States), and stable RCC1-expressing cells were selected via G418 screening (named Huh-7-RCC1 and HCC-LM3-RCC1). pcDNA3.1-RCC1 plasmid was procured from Tsingke Biotech Co., Ltd. (Beijing, China). The RCC1 and CDCA8 small interfering RNAs (siRNAs) were synthesized by Tsingke Biotech Co., Ltd. (Beijing, China). According to the manufacturer’s instructions, plasmids or siRNA were transfected into cell lines using Lipofectamine 2000. The mRNA levels in transiently transfected cells were assessed 24 hours post-transfection, and protein levels were measured 48 hours post-transfection. The siRNA sequences are listed in Table 1.

Table 1 Sequences of small interfering RNAs.

siRNAs	Sense/antisense	Sequences
siRCC1	Sense	5’-GGCACAGAAUCUUGCUUCAUATT-3’
	Antisense	5’-UAUGAAGCAAGAUUCUGUGCCTT-3’
siCDCA8	Sense	5’-GGAAAUACGAAUCAAGCAATT-3’
	Antisense	5’-UUGCUUGAUUCGUAUUUCCTT-3’

siRNA: Small interfering RNA; siRCC1: Small interfering regulator of chromosome condensation 1; siCDCA8: Small interfering cell division cycle-associated 8.

Real-time quantitative reverse transcription polymerase chain reaction

Total RNA was extracted using the Total RNA Kit II (Omega, Riverside, CA, United States) and reverse transcribed into cDNA via the PrimeScript RT reagent kit (TaKaRa Biotechnology, Otsu, Japan). Then the single-stranded cDNA was amplified by quantitative reverse transcription polymerase chain reaction using TB Green Premix Ex Taq II (TaKaRa Biotechnology) on a Stratagene Mx3005P real-time polymerase chain reaction System (Agilent Technologies, Santa Clara, CA, United States). The primer sequences are listed in Table 2.

Table 2 Primer sequences used in real time quantitative reverse transcription polymerase chain reaction.

Gene symbol	Forward/reverse	Sequences
RCC1	Forward	5’-ACTCCGGGCACAGAATCTTG-3’
	Reverse	5’-CCTGGAGATGAGGGTGGGTA-3’
CDCA8	Forward	5’-GAAGCTCGCCTCCTTTCTGA-3’
	Reverse	5’-CTCTTCCAGGGCCTGTTTGT-3’
MCM4	Forward	5’-CCTGGTCGCACTGTACTACC-3’
	Reverse	5’-AAACCATTCCCCGGCTACTG-3’
CCNB1	Forward	5’-TGTGTGCCCAAGAAGATGCT-3’
	Reverse	5’-AAGTGCAAAGGTAGAGGCCG-3’
CDCA5	Forward	5’-CTCTGAACTCCCGAGCATCC-3’
	Reverse	5’-GATTGGACAGCTGGGACCTC-3’
GAPDH	Forward	5’-CCAGAACATCATCCCTGCCT -3’
	Reverse	5’-CCTGCTTCACCACCTTCTTG -3’

Immunoblotting analysis

Cells were lysed on ice with RIPA Lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1 mL/L Nonidet P-40, 2 mmol/L EDTA and protease inhibitors) for 20 minutes, followed by centrifugation at 12000 rpm for 20 minutes at 4 °C. The supernatant was collected, and protein concentrations were determined using the bicinchoninic acid assay. Add appropriate volume of 5 × loading buffer to the protein samples, heat the mixture in a metal bath at 100 °C for 10 minutes. Equal amounts of protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 150 V for 1 hour and transferred to a polyvinylidene difluoride membrane on ice at 100 V for 1 hour. The membrane was blocked with 50 g/L skimmed milk in Tris-buffered saline with Tween 20 at room temperature for 1 hour, followed by incubation with the primary antibody in Tris-buffered saline with Tween at 4 °C overnight. Secondary antibodies used included horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG), HRP-goat anti-rabbit IgG and HRP-rabbit anti-goat IgG (Pierce, Rockford, IL, United States). The reactive bands were detected using the Western-Light chemiluminescent detection system (Image Station 4000 MM Pro, XLS180, Kodak, Rochester, NY, United States).

Antibodies and reagents

The anti-β-actin (66009-1-Ig, western blot (WB): 1:10000), anti- RCC1 (22142-1-AP, WB: 1:2000), anti-CDCA8 (12465-1-AP, WB: 1:1000), anti-minichromosome maintenance complex component (MCM) 4 (13043-1-AP, WB: 1:1000), anti-CDCA5 (67418-1-AP, WB: 1:3000), anti-phosphatidylinositol - 4,5 - bisphosphate 3 - kinase catalytic subunit alpha (PIK3CA) (67071-1-AP, WB: 1:2000), anti-Akt (60203-2-Ig, WB: 1:10000) and anti-Phospho-Akt (Ser473) (66444-1-AP, WB: 1:5000) antibodies were purchased from Proteintech (Wuhan, China). Anti-cyclin B1 (CCNB1) (12231, WB: 1:1000) was obtained from Cell Signaling Technology (Boston, MA, United States). Anti-p21 (ab109520, WB: 1:5000) was purchased from Abcam (Cambridge, United Kingdom). The Enhanced Cell Counting Kit-8 (CCK-8) (C0042) and BeyoClick^™ EdU Cell Proliferation Kit with Alexa Fluor 555 (C0075S) were purchased from Beyotime (Shanghai, China). The Cell Cycle Detection Kit (KGA9101-100) was purchased from Keygen Biotech (Jiangsu Province, China).

Co-immunoprecipitation

The interaction between RCC1 and CDCA8 in HCC cells was detected using a co-immunoprecipitation (IP) kit (Pierce, 26149) according to the manufacturer’s instructions. Anti-RCC1 or anti-CDCA8 antibodies were introduced to the resin and incubated overnight at 4 °C. Subsequently, the IP lysate was applied to the cells and incubated on ice for 5 minutes to facilitate cell lysis. The resultant lysate was then transferred to the resin and subjected to overnight incubation at 4 °C. Finally, 50 μL of eluent was added, followed by a 5 minutes incubation at room temperature, and the filtrate was collected via centrifugation. The samples were then analyzed by immunoblotting.

CCK-8 assay

Cells (2000 per well) were seeded into a 96-well plate with 100 μL of cell suspension per well. The cells were incubated at 37 °C overnight for adherence. At designated time intervals (0, 12, 24, 36, 48, 60, 72 hours), 10 μL of CCK-8 solution was added to each well. After an additional hour of incubation, absorbance at 450 nm was measured using a microplate reader.

5-Ethynyl-2’-deoxyuridine incorporation assay

Cells were cultured in a dish of 12 hours, followed by treatment with 10 μmol/L 5-ethynyl-2’-deoxyuridine (EdU) for 2 hours at 37 °C. Cells were fixed with 40 g/L paraformaldehyde at room temperature for 15 minutes. After fixation, cells were washed three times with phosphate-buffered saline (PBS) containing 30 g/L BSA for 5 minutes. Cells were then incubated in PBS with 3 mL/L Triton X-100 at room temperature for 15 minutes. Following this, the EdU detection solution was added, and the cells were incubated at room temperature in the dark for 30 minutes. Cell nuclei were stained with Hoechst 33342 and incubated in the dark at room temperature for 10 minutes before imaging.

Cell cycle analysis

Harvest cells and fix them in 1 mL of pre-cooled 700 mL/L ethanol absolute. Gently pipette to ensure thorough mixing and allow the cells to fix overnight at 4 °C. Centrifuge the cells at 1000 r/minute for 5 minutes to precipitation. Decant the supernatant, add 1 mL of pre-cooled PBS, and resuspend the cells. After a second centrifugation, remove the supernatant and add 0.5 mL of PI Staining solution to each tube. Incubate the samples at room temperature in the dark for 1 hour, followed by analysis using flow cytometry.

Wound healing assay

Monolayer cells in culture plates were scraped using a 100-μL pipette tip. Images were captured at different time points (0 hour and 24 hours) post-scratching. The width of the scratch was measured to ensure consistency at the initial time point.

Transwell invasion assay

Add the matrix gel diluted with serum-free medium to the upper surface of the bottom membrane within the Transwell chambers. Incubate for 2 hours at 37 °C to facilitate the polymerization of the matrix gel into a gel film. Subsequently, remove any excess liquid and add 100 μL of serum-free medium to each well. Incubate at 37 °C for an additional 30 minutes to achieve hydration of the basement membrane. Next, Transwell chambers were placed in 24-well cell culture plate, with 200 μL of cell suspension (devoid of FBS) added to the upper chamber and 600 μL of culture medium containing 100 mL/L FBS added to the lower chamber for a duration of 24 hours. Subsequently, the cells that had migrated to the bottom were fixed with 40 g/L paraformaldehyde and stained with 2 g/L crystal violet for imaging.

Immunohistochemistry

Tissues were deparaffinized using xylene and alcohol, followed by antigen retrieval with 1 mol/L Citrate Buffer (pH 6.0) (ZLI-9065, Zhongshan Jinqiao Co., Beijing, China) for 2 minutes at 120 °C. Subsequently, the tissues were treated with 3 g/L H₂O₂ and blocked with normal goat serum. The samples were then incubated with primary antibodies at 4 °C overnight in a humidified chamber. Post-incubation, immunoperoxidase staining was performed utilizing a streptavidin-peroxidase kit (Zhongshan Jinqiao Co., Beijing, China) and 3,3’-diaminobenzidine (Zhongshan Jinqiao Co., Beijing, China) was employed to visualize the target proteins.

Statistical analysis

Data obtained from at least three independent experiments were expressed as means ± SEM and analyzed using GraphPad Prism v8.0 software (GraphPad Software, LaJolla, CA, United States). One-way analysis of variance and t-tests were conducted to compare mean values. Student’s t-test was conducted to compare two individual data. analysis of variance was employed to assess the significance of differences among the various groups. Statistical significance was set at P < 0.05.

RESULTS

RCC1 is highly expressed in HCC and correlates with a poor prognosis in HCC patients

Elevated levels of RCC1 expression have been documented in various cancer types[32]. Analysis of data from TCGA and International Cancer Genome Consortium revealed that RCC1 expression in HCC samples were significantly higher than in adjacent tissue samples (Figure 1A). To further investigate the expression of RCC1 in HCC, we compared its levels between the murine normal hepatic cell line AML12 and the murine hepatic cancer cell line Hepa1-6. Our results indicated that RCC1 expression was significantly elevated in the Hepa1-6 cell line compared to the AML12 cell line (Supplementary Figure 1A and B). To validate these findings in human tissues, we performed immunoblotting analysis on six paired human HCC samples and adjacent non-cancerous tissues and immunohistochemical staining on ten paired human HCC samples and adjacent non-cancerous tissues. Consistently, RCC1 protein levels were markedly higher in HCC tissues than in adjacent normal tissues (Supplementary Figure 1C-F), aligning with our previous bioinformatics analysis (Figure 1A). These results collectively suggest that RCC1 overexpression is associated with HCC progression across species. Next, the HCC samples were stratified into RCC1-high and RCC1-low groups based on the median RCC1 expression level. Kaplan-Meier survival analysis indicated that elevated RCC1 expression was associated with reduced overall survival in HCC patients (Figure 1B). Furthermore, a statistically significant positive correlation was observed between elevated RCC1 expression and advanced pathologic grade (Figure 1C).

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.

We next conducted a differential gene expression analysis comparing the RCC1-high group to the RCC1-low group, identifying 226 up-regulated genes and 53 down-regulated genes (Figure 1D). Herein, we present the 50 most significantly up-regulated and 50 most significantly down-regulated genes (Figure 1E). Given the predominance of up-regulated genes, we performed functional enrichment analyses on these differentially up-regulated genes, including GO term enrichment (Figure 1F) and KEGG pathway enrichment (Figure 1G). The KEGG pathway enrichment analysis revealed significant enrichment in the cell cycle and DNA replication pathways while the GO term enrichment analysis demonstrated an enrichment of biological processes, including mitosis, DNA replication, and cell cycle checkpoints. Subsequently, we employed Gene Set Enrichment Analysis to further investigate the association between RCC1 expression and both DNA replication and tumor proliferation, revealing a robust positive correlation between RCC1 expression and these biological processes (Figure 1H). These findings suggest that RCC1 exerts a pro-oncogenic influence in HCC, potentially contributing to the unfavorable prognosis observed in HCC patients. It is posited that RCC1 may participate in the regulation of the cell cycle and cell proliferation, thereby facilitating the development of HCC.

RCC1 facilitates the proliferation of HCC cells

To elucidate the impact of RCC1 on HCC cells, we established HCC cell lines (Huh-7-RCC1 and HCC-LM3-RCC1) with stable overexpression of RCC1 (Figure 2A and B, Supplementary Figure 2A and B) and other cell lines (Huh-7-siRCC1 and HCC-LM3-siRCC1) with RCC1 knockdown using siRNA (Figure 3A and B, Supplementary Figure 3A and B). CCK-8 assays showed that RCC1 overexpression significantly enhanced the viability of HCC cells (Figure 2C, Supplementary Figure 2C), while RCC1 knockdown in HCC cells inhibited cell growth (Figure 3C, Supplementary Figure 3C). EdU incorporation assays further confirmed that RCC1 enhances HCC cells proliferation (Figure 2D and E, Figure 3D and E, Supplementary Figure 2D and E, Supplementary Figure 3D and E). To determine whether the observed proliferation effect is attributable to cell cycle influence, we employed flow cytometry to assess alterations in cell cycle distribution. The findings indicated that RCC1 overexpression increased the proportion of cells in the S and G₂/M phases, and decreased the proportion in the G₀/G₁ phase (Figure 2F and G, Supplementary Figure 2F and G), while RCC1 silencing hindered the transition of HCC cells from the G₁ to the S phase (Figure 3F and G, Supplementary Figure 3F and G). In addition to its role in cell proliferation, we also found that RCC1 promoted the migration and invasion potential in both Huh-7 and HCC-LM3 cells (Figure 2H-K, Figure 3H-K, Supplementary Figure 2H-K, Supplementary Figure 3H-K). Collectively, these findings suggest that RCC1 might critically influence HCC progression through regulating cellular behaviors such as proliferation.

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.

RCC1 modulates the expression of cell cycle-associated genes in HCC

To investigate the regulatory role of RCC1 in the cell cycle of HCC, we used The University of Alabama at Birmingham CANcer data analysis Portal to identify genes correlated with RCC1 in HCC samples from the TCGA database (Figure 4A). Enrichment analysis was performed on genes with correlation coefficients exceeding 0.5, revealing an enrichment of pathways and processes related to the cell cycle and DNA replication (Figure 4B). These findings supported previous bioinformatics analyses (Figure 1). We identified cell cycle-associated genes from the top ten genes with significant correlation coefficients, specifically CDCA8, MCM4, CCNB1, and CDCA5 (Figure 4A). We then evaluated the expression changes of these genes in Huh-7-RCC1 and Huh-7-siRCC1 cell lines. The results showed that RCC1 overexpression increased both mRNA and protein levels of CDCA8, MCM4, CCNB1, and CDCA5 (Figure 4C-E), while RCC1 knockdown decreased these levels (Figure 4F-H). These findings suggest that RCC1 modulates cell cycle progression in HCC by regulating the expression of cell cycle-related genes.

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.

RCC1 regulates CDCA8 to enhance cell proliferation in HCC

To clarify the downstream target genes influenced by RCC1, we analyzed the expression correlation between RCC1 and CDCA8, MCM4, CCNB1, and CDCA5 (Figure 5A). Our findings indicated that the correlation between RCC1 and CDCA8 was the most significant. Furthermore, differential gene analysis comparing the high and low RCC1 expression groups revealed that CDCA8 was the most significantly upregulated gene (Figure 1E). Consequently, our subsequent research focused on CDCA8. Initially, we employed RCC1 and CDCA8 antibodies for co-IP assays, respectively. The results demonstrated a distinct interaction between RCC1 and CDCA8 (Figure 5B). Subsequently, CDCA8 was silenced in the Huh-7-RCC1 cell line, followed by a series of functional assays. CCK-8 assays showed that RCC1 overexpression enhanced HCC cell proliferation, whereas concurrent CDCA8 interference inhibited HCC cell growth, with no significant changes observed in the control group (Figure 5C). These findings were corroborated by the EdU incorporation assay, which yielded consistent results (Figure 5D and E). Next, cell cycle analysis revealed that RCC1 overexpression increased the proportion of HCC cells in the S and G₂/M phase. Conversely, concurrent interference with CDCA8 resulted in a reduction of cells in these phases (Figure 5F and G). Otherwise, the results of Transwell invasion assay and wound healing assay indicated that disrupting CDCA8 can suppress RCC1-induced invasion and migration of HCC cells (Supplementary Figure 4A-D). In conclusion, these findings collectively indicate that RCC1 exerts its tumor-promoting function in HCC development through a CDCA8-dependent regulatory mechanism.

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.

RCC1 activates PI3K/Akt/p21 pathway through CDCA8 to promote HCC proliferation

Previous research has shown that CDCA8 can activate the PI3K/Akt signaling pathway, thereby promoting the proliferation and migration of melanoma cells[33]. This raises the question of whether the RCC1/CDCA8 axis similarly facilitates the HCC proliferation through the regulation of the PI3K/Akt pathway. To test this hypothesis, we analyzed the expression levels of PIK3CA, p-Akt, and p21. Our findings indicated that RCC1 overexpression enhanced the expression of PIK3CA and p-Akt while inhibiting p21 expression (Figure 6A and B), whereas RCC1 silencing had the opposite effects (Figure 6C and D). These results suggested a significant correlation between RCC1 and the PI3K/Akt signaling pathway. Subsequently, it was crucial to determine whether RCC1 modulated the PI3K/Akt signaling pathway through CDCA8. To address this, we assessed the protein expression levels of p-Akt, PIK3CA, and p21 in Huh-7-RCC1 cells following CDCA8 knockdown (Figure 6E). The findings showed that RCC1 overexpression led to increased levels of p-Akt and PIK3CA proteins, and decreased p21 protein levels. Conversely, upon CDCA8 knockdown, there was a marked decrease in p-Akt and PIK3CA protein levels, accompanied by a significant increase in p21 protein levels (Figure 6F). These results suggest that RCC1 regulates the PI3K/Akt/p21 signaling pathway through CDCA8, thereby promoting cell proliferation in HCC.

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.

DISCUSSION

In this study, we investigated the role of RCC1 in promoting the proliferation of HCC cells. Initially, we determined that RCC1 is significantly upregulated in HCC and correlates with poor prognosis. Subsequently, we demonstrated that RCC1 facilitates the G₁/S phase transition by regulating CDCA8. Furthermore, our findings indicate that the RCC1/CDCA8 axis promotes HCC proliferation via the PI3K/Akt/p21 signaling pathway. Collectively, this research elucidates the association between RCC1 and HCC and highlights the role of RCC1 in regulating the G₁/S transition of the cell cycle.

Abnormal cellular proliferation is a critical factor in the initiation and progression of tumors[34]. RCC1 functions as a guanine nucleotide exchange factor for the Ras-like G protein Ran within the nucleus and is integral to the G₁/S transition of the cell cycle[35]. RCC1 overexpression leads to elevated levels of Ran-GTP, which in turn accelerates the cell cycle and enhances DNA damage repair mechanisms, thereby facilitating tumor progression[8]. Specifically, in non-small cell lung cancer, the knockdown of RCC1 has been shown to inhibit the proliferation of lung adenocarcinoma cells and decelerate tumor growth[36]. Similarly, in renal clear cell carcinoma, RCC1 promotes cell proliferation by accelerating the cell cycle and inhibiting apoptosis[37]. Furthermore, RCC1 knockdown markedly impedes the cell cycle transition, proliferation, invasion, and migration of soft tissue sarcoma cells, in addition to suppressing the growth of soft tissue sarcoma xenograft tumors in murine models[11]. However, it remains uncertain if RCC1 promotes the development of HCC as observed in other cancers. This study demonstrates that RCC1 expression is elevated in HCC and exhibits a significant positive correlation with biological processes, including the cell cycle and DNA replication. RCC1 overexpression significantly promoted HCC proliferation by increasing the proportion of cells in S and G₂/M phases, while concomitantly enhancing cell migration and invasion. Conversely, interference with RCC1 expression inhibited the G₁/S transition and the cell growth of HCC. In summary, RCC1 plays a critical role in promoting cell proliferation in HCC.

Aberrant expression of cyclins and cyclins associated proteins is a characteristic feature of cancer development[38]. Our further studies have revealed that RCC1 regulates the expression of several cyclins and cyclins associated proteins in HCC, including CDCA8, MCM4, CCNB1, and CDCA5. Notably, CDCA8 is identified as an oncogene that is upregulated across multiple cancer types and is crucial for the survival and progression of various cancer cells[14]. Research indicates that CDCA8 is highly expressed in breast cancer cells and serves as a key mediator of estrogen-induced proliferation and survival[20]. Furthermore, the synergistic interaction between CDCA8 and E2F transcription factor 1 enhances the glioma malignancy by facilitating cell proliferation and migration while inhibiting apoptosis[39]. In the context of HCC, the silencing of CDCA8 results in the downregulation of CDK1 and CCNB1, thereby inhibiting cell proliferation and promoting apoptosis[13], findings that align with our results. Our research has demonstrated that the knockdown of CDCA8 impedes RCC1-mediated cell growth and the G₁/S phase transition of the cell cycle. In conclusion, RCC1 modulates CDCA8 to promote the proliferation of HCC cells.

The PI3K/Akt/p21 signaling pathway, mediated by Akt phosphorylation and activation, is frequently hyperactivated in numerous tumor types, facilitating cellular proliferation and migration[40-42]. Furthermore, prior research has elucidated the association between CDCA8 and the PI3K/Akt signaling pathway. For instance, CDCA8 has been recognized as a pivotal gene in osteosarcoma, with CDCA8 and other core genes predominantly enriched within the PI3K/Akt signaling pathway[43]. Additionally, another study demonstrated that TMED3 enhances melanoma progression by modulating the PI3K/Akt pathway through CDCA8[33]. Interestingly, RCC1 is also associated with the PI3K/Akt pathway. Phosphorylation modification at S11 site of RCC1 can regulate the cell cycle through the PI3K/Akt signaling pathway, affecting the occurrence and development of cervical cancer[44]. Consequently, we investigated the potential role of the RCC1/CDCA8 axis in facilitating the progression of HCC via the PI3K/Akt/p21 signaling pathway. In RCC1-overexpressing cells with CDCA8 silencing, we quantified the expression levels of p-Akt, PI3KCA, and p21. Our findings revealed a reduction in p-Akt and PI3KCA levels, alongside elevated in p21 Levels. These results suggest that RCC1 may enhance HCC development by modulating the PI3K/Akt/p21 pathway through CDCA8.

CONCLUSION

In summary, our study has provided profound insights into the pivotal role of RCC1 in HCC and its potential as a therapeutic target. The overexpression of RCC1 is prevalent in HCC and contributes to the proliferation and survival of tumor cells by modulating CDCA8 to activate the PI3K/Akt/p21 signaling pathway. This finding not only elucidates the significance of RCC1 in the initiation and progression of HCC but also offers novel perspectives for future HCC treatment. Targeting the RCC1/CDCA8 axis and its associated signaling pathways holds promise as an effective anticancer strategy. Moreover, the combination with PI3K/Akt pathway inhibitors may further enhance therapeutic efficacy, bringing more hope to HCC patients.