Open Access

CSN1 facilitates proliferation and migration of hepatocellular carcinoma cells by upregulating cyclin A2 expression

  • Authors:
    • Hangwei Fu
    • Yida Zhang
    • Yin Chen
    • Junying Chen
    • Ping Chen
  • View Affiliations

  • Published online on: November 12, 2020     https://doi.org/10.3892/mmr.2020.11684
  • Article Number: 46
  • Copyright: © Fu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Constitutive photomorphogenesis 9 signalosome subunit 1 (CSN1) plays an important role in the ubiquitin-proteasome pathway and regulates various cellular processes, such as the cell cycle and DNA repair. The CSN complex consists of eight subunits (CSN1 to CSN8) and regulates the tumorigenesis of a variety of tumor types. However, the exact role of CSN1 in hepatocellular carcinoma (HCC) remains unclear. The present study evaluated the expression and biological effects of CSN1 in HCC tissue samples and cell lines. CSN1 was significantly overexpressed in HCC tissue and cell lines, compared with their normal counterparts. In patients with HCC, elevated CSN1 levels correlated with tumor size, tumor metastasis and tumor stage. Loss‑of‑function assays indicated that CSN1 knockdown inhibited the proliferation and migration HCC cells. In addition, CSN1 promoted the expression of cyclin A2 in a ubiquitination‑independent manner. Lastly, xenograft experiments indicated that CSN1 promoted HCC tumor growth in vivo. The present study suggested that CSN1 inhibition could represent a potential approach for the prevention of HCC progression and metastasis.

Introduction

Hepatocellular carcinoma (HCC) has the sixth highest incidence among all malignant tumors worldwide and the fourth highest mortality rate worldwide (1). More than half of all patients with HCC worldwide reside in China (2). In recent years, the incidence of liver cancer has been on the rise, yet <20% of patients receive timely radical surgical resection, due to the high degree of malignancy, early metastasis and high invasion potential of this tumor type. These properties are often associated with the complex biochemical regulatory behavior of HCC cells, such as excessive cell proliferation (3,4). Although HCC treatment methods and efficiency have improved the 5-year survival rate, several methods such as radical resection, transcatheter arterial chemoembolization, sorafenib and chemoradiotherapy still have their respective limits (5). Radical hepatectomy is considered the first choice for the treatment of early HCC at an early stage (Barcelona Clinic Liver Cancer stage 0 or A). For these patients, resection is associated with survival >60% at 5 years, with low postoperative mortality (<3%), and as many as 70% of these patients experience tumor recurrence at 5 years (6). Based on HCC pathogenesis, in addition to a number of correlative studies, immunotherapy represents a potential therapeutic option (7). Therefore, investigating the mechanism underlying the onset of HCC and finding a potential therapeutic target is of significance for improving the diagnosis and treatment of HCC.

Constitutive photomorphogenesis 9 signalosome (COP9) is a protein complex composed of eight subunits (CSN1 to CSN8) that was initially discovered in plants as an inhibitor of photomorphogenesis (812). Mammalian COP9 is involved in a variety of biological processes, including cell cycle control, signal transduction, transcriptional activation and tumorigenesis (1315). CSN subunits have a conserved structure; for example, the Mpr1-Pad1-N-terminal (MPN) domain in CSN5 contains a metalloproteinase motif, which has catalytic activity and is involved in the regulation of protein demethylation (1618). MPN in CSN6 may serve as a structural scaffold or play a regulatory function (16,19,20). Previous studies have confirmed that CSN inhibits the ubiquitin-dependent degradation of numerous tumor-related proteins (2123), such as P27, P53, E3 ubiquitin-protein ligase Mdm2, Smad7, Runt-related transcription factor 3, DNA-binding protein inhibitor ID-1, S-phase kinase-associated protein 2 and hypoxia-inducible factor 1 (14), indicating that CSN plays an important role in tumorigenesis. In particular, CSN5 or CSN6 overexpression has been detected in numerous types of cancer, such as myeloma, lung cancer and breast cancer (24,25). In HCC, CSN6 overexpression promotes epithelial-mesenchymal transition and predicts poor prognosis (26). Inhibition of CSN3 expression induced growth arrest and apoptosis of HCC cells (27). In our preliminary study, by analyzing the transcriptome data of patients within The Cancer Genome Atlas, it was found that CSN1 expression was increased in patients with HCC compared with normal controls. However, the role and molecular mechanism of CSN1 in HCC remains to be elucidated.

Cyclin A2 is a highly conserved cell cycle protein encoded by the CCNA2 gene. Cyclin A2, combined with cyclin-dependent kinase 1 (CDK1) and CDK2, controls cell cycle progression and promotes mitosis (28) and serves a potential role in liver tumorigenesis (29). Cyclin A2 has been recognized as a marker for several types of cancer diagnosis and prognosis (30), such as lung carcinoma, breast cancer, hepatocellular carcinoma and colorectal cancer. Moreover, the expression of cyclin A2 is increased in HCC (31), indicating that this protein may play an important role in the development of HCC and represent a potential therapeutic target.

In the present study, CSN1 was upregulated in HCC tissue samples, compared with healthy adjacent tissue. In addition, high levels of CSN1 indicated poor prognosis in patients with HCC. CSN1 knockdown inhibited proliferation and migration and induced apoptosis and cell cycle arrest in HCC cells. Moreover, xenograft tumor experiments demonstrated that CSN1 promoted HCC in vivo. Mechanistically, cyclin A2 may be a downstream effector of CSN1.

Materials and methods

Patients and sample collection

Primary HCC tissue samples were obtained for retrospective analysis from 117 patients at Daping Hospital, Army Medical University, between January 2013 and December 2014. Matched, adjacent, non-tumor tissue samples resected 1–2 cm from the malignant tumor were obtained from 59 of these patients. Written informed consent was obtained from all participants. All specimens obtained during surgery were assembled into a tissue microarray (TMA) for further analysis. The identification of tumor tissues and adjacent normal tissues was confirmed by pathologists. The present study was approved by the Institutional Research Ethics Committee of Daping Hospital, Army Medical University.

Cell culture and transfection

Human HCC cell lines (MHCC-97H, MHCC-LM3 and Hep3B) were purchased from the Cell Bank of Academia Sinica. The immortalized liver cell line MIHA was a kind gift from Dr Deng Huang. The cells were cultured in DMEM (HyClone; Cytiva) supplemented with 10% FBS (Shanghai ExCell Biology, Inc.) at 37°C with 5% CO2.

Human CSN1 and cyclin A2 small interfering (si) RNA molecules were synthesized by Shanghai GenePharma Co., Ltd. The siRNA sequences were as follows: i) si-CSN1-1, 5′-CUGCCGGUUCAGGUGUUATT-3′; ii) si-CSN1-2, 5′-GAACCUUUAACGUGGACAUTT-3′; iii) si-CSN1-3, 5′-GAGACAUCAUCUUCAAAUUTT-3′; iv) si-cyclin A2, 5′-CAACCCACCAGAGACACUAAATT-3′; and v) si-negative control (NC), 5′-UUCUCCGAACGUGUCACGUTT-3′. Human CSN1 overexpression (OE) plasmid and its corresponding empty vector (pReceiver-M02) were purchased from GeneCopoeia, Inc. Plasmids (2 µg) or siRNA (50 nM) were transfected using Lipofectamine® 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Subsequent cell experiments were conducted after transfection for 48 h.

The 293T cells were inoculated in 10-cm culture plates at a density of 5×106 cells in 15 ml and allowed to reach 70–80% confluence the day prior to infection. The lentiviral plasmid GV (20 µg), and packaging plasmids pHelper 1.0 (15 µg) and pHelper 2.0 (10 µg) (Shanghai GeneChem Co., Ltd.) were transfected into 293T cells using Genechem Transfection Reagent (Shanghai GeneChem Co., Ltd.) for 6 h at 37°C in 5% CO2, according to the manufacturer's protocol. Following 48–72 h, supernatants containing lentiviral particles were harvested and filtered through a 0.45-µm filter (EMD Millipore) to remove cell debris. The supernatants were concentrated by ultracentrifugation at 25,000 × rpm at 4°C for 2 h, and the lentiviral particle pellet was resuspended in 100% FBS and stored at −80°C. The viral titers of concentrated lentiviral particles were measured by infecting 293T cells seeded at a density of 4×104 cells/well in a 96-well plate with viral serial dilutions. Green fluorescent protein (GFP) expression was detected 4 days later under a fluorescence microscope and the viral titer was calculated using the following equation: Viral titer (Tu/µl)=(% GFP+ cells × number of cells transduced)/virus volume.

For lentiviral transduction, MHCC-LM3 were seeded at 1×105 cells/ml in 24-well plates, and the concentrated lentivirus was added at MOI=20 at 37°C and 5% CO2. Seven days after transfection, 1 µg/ml puromycin (cat. no. ST551; Beyotime Institute of Biotechnology) was added into the cell culture medium to select the transduced cells.

Western blot analysis

Total protein was extracted from cells using RIPA lysis buffer (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Total protein was quantified using a BCA Protein Assay kit (cat. no. P0012; Beyotime Institute of Biotechnology) and equivalent amount of 20 µg protein samples were separated with a 4–12% Bis-Tris gel (Beyotime Institute of Biotechnology) and transferred onto a PVDF membrane. The membrane was blocked with 5% non-fat milk in TBS-Tween-20 for 1 h at room temperature, incubated with the indicated primary antibodies overnight at 4°C, and then with horseradish peroxidase-conjugated secondary antibodies (1:5,000; ProteinTech Group, Inc.) at room temperature for 1 h. The primary antibodies used were specific for CSN1 (1:2,000; cat. no. 11709-1-AP; ProteinTech Group, Inc.), cyclin A2 (1:2,000, cat. no. GTX103042; GeneTex, Inc.), CDK-2 (1:2,000; cat. no. 10122-1-AP; ProteinTech Group, Inc.), CDK-4 (1:2,000; cat. no. 11026-1-AP; ProteinTech Group, Inc.), phospho (p)-CDK4 (1:2,000, cat. no. GTX00778; GeneTex, Inc.) or GAPDH (1:5,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.). HRP-conjugated Affinipure goat anti-mouse IgG (H+L) (1:5,000; cat. no. SA00001-1; ProteinTech Group, Inc.) and HRP-conjugated AffiniPure goat anti-rabbit IgG (H+L) (1:5,000; cat. no. SA00001-2; ProteinTech Group, Inc.) were used as secondary antibodies. Protein signals were detected by enhanced chemiluminescence (cat. no. KGP1121; Nanjing KeyGen Biotech Co., Ltd.). Data were semi-quantified using ImageJ software (v1.48; National institutes of Health). GAPDH was used as internal control.

Immunohistochemistry for TMA

TMAs were constructed from formalin-fixed, paraffin-embedded tissue blocks stored at the Department of Pathology, Daping Hospital. All specimens were previously fixed in 10% neutral formalin solution at room temperature for 12 h before being paraffin-embedded and cut to 8-µm thick sections. Following incubation at 65°C for 1 h, TMA slides were dewaxed with dimethylbenzene and then rehydrated with graded alcohol and distilled water. For antigen retrieval, TMA slides were incubated in a microwave oven heated at 95°C with 10 mM citrate buffer (pH 6.0). Endogenous peroxidases were quenched with 3% H2O2 for 20 min. The slides were treated with normal goat serum (cat. no. C0265; Beyotime Institute of Biotechnology) to inhibit non-specific staining at room temperature for 15 min. Subsequently, the sections were incubated with anti-CSN1 antibody (1:50; cat. no. 11709-1-AP; ProteinTech Group, Inc.) overnight at 4°C and then incubated with goat anti-rabbit antibody (1:500; cat. no. C0265; Beyotime Institute of Biotechnology) for 1 h. Diaminobenzidine (OriGene Technologies, Inc.) was used to produce a brown staining. Once hematoxylin counterstaining and dehydration were completed, the sections were sealed. Immunohistochemical staining of the TMA was assessed by light microscopy. Three random fields of view of the IHC chips were captured. The expression levels of CSN1 were quantified by counting mean gray value with ImageJ (version 1.48; National Institutes of Health). A value higher than the mean was defined as high expression, while a value equal to or lower than the mean was categorized as low expression in tumors.

Cell proliferation assay

HCC cells were seeded into 96-well plates at a density of 5×103 cells/well and transfected with the indicated siRNA or plasmids. At 48 h after transfection, cell proliferation was determined in vitro using the EdU DNA Proliferation Detection kit (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's instructions.

Transwell cell migration and wound healing assays

Transwell chambers (BD Biosciences) with a pore size of 8 µm were used to determine cell migration. MHCC-97H or MHCC-LM3 cells (2×104) were seeded in the upper chamber in serum-free DMEM (HyClone; Cytiva). Medium containing 10% FBS was added to the lower chamber. Following incubation at 37°C for 24 h, the cells were fixed in methanol and stained with leucocrystal violet at room temperature for 10 min. Cells in the upper chamber were removed, and the number of cells that migrated across the membrane was determined by counting the number of leucocrystal violet-stained cells. Stained cells were examined under a light microscope and quantified by counting all the cells in the field of view. At least three random microscopic fields were captured.

MHCC-97H or MHCC-LM3 cells (3×105 cells/well) were seeded and transfected as aforementioned in 6-well plates. The cells were then scratched with a 10-µl pipette tip along the center of the plate when the cells reached ~95% confluence. The HCC cells were incubated in serum-free medium. The distances between the cells bordering the wound were measured and analyzed under a light microscope at 0, 24 and 48 h.

Cell cycle and cell apoptosis analysis

For cell cycle and cell apoptosis analysis, MHCC-97H or MHCC-LM3 cells were seeded at a density of 3×105 cells/well in a 6-well plate and transfected with the indicated constructs. At 48 h after transfection, the cells were harvested by centrifugation at 1,000 × g for 5 min and fixed in 70% ethanol overnight at 4°C. The cells were then analyzed using DNA Content Quantitation Assay (Cell Cycle) (cat. no. CA1510-50T; Beijing Solarbio Science & Technology Co., Ltd.) according the manufacturer's protocol.

The apoptosis rate of HCC cells was analyzed using an Annexin V-FITC/PI apoptosis detection kit (cat.no. CA1020-50T; Beijing Solarbio Science & Technology Co., Ltd.) following the manufacturer's instructions. In brief, 5×105 cells were harvested by centrifugation at 1,000 × g for 5 min and resuspended in 200 µl binding buffer, followed by a 15-min incubation with 5 µl Annexin V-FITC and 5 µl propidium iodide in the dark at 37°C. Data were acquired using a FACSVerse flow cytometer (BD Biosciences) and analyzed using FlowJo software (10.0.7; FlowJo LLC). Annexin V+PI cells were identified as early apoptotic cells, whereas Annexin V+PI+ cells were considered late apoptotic cells.

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was isolated from cells using the TRIpure total RNA extraction kit (cat. no. RP1001; BioTeke Corporation). RT was performed using the PrimeScript™ RT Reagent kit, and qPCR was performed using SYBR Premix Ex Taq II (both from Takara Bio, Inc.) according to the manufacturer's instructions. RT was carried out at 37°C for 15 min then 85°C for 15 sec. qPCR was performed at 95°C for 30 sec, followed by 40 cycles of 5 sec at 95°C and 30 sec at 60°C. GAPDH was used as reference gene. The following primer pairs were used: GAPDH forward, 5′-ACTCCTCCACCTTTGACGC-3′ and reverse, 5′-GCTGTAGCCAAATTCGTTGTC-3′; cyclin A2 forward, 5′-CCAGGAGAATATCAACCCGGA-3′ and reverse, 5′-GTGCAACCCGTCTCGTC-3′ The relative expression levels of each target were quantified using the 2−ΔΔCq method (32).

Protein half-life assay

To examine the half-life of cyclin A2 protein, cycloheximide pulse-chase experiments were performed as previously described (33). MHCC-LM3 cells were seeded at 3×105 cells/well in 6-well plates. Twenty-four hours later, the cells were treated with 75 µg/ml cycloheximide (CHX; Sigma-Aldrich; Merck KGaA) to block the endogenous protein synthesis for 0, 1, 2, 4, 6 and 8 h, and were subsequently lysed for western blotting. The bands were scanned, and the image semi-quantified using ImageJ software (v1.48; National institutes of Health). The relative concentration at 0 h was defined as 100%. The fraction of remaining cyclin A2 at each indicated time point was normalized by comparing the relative concentration with that at time 0 h.

Protein synthesis inhibition assay

CHX or actinomycin D were used to block the endogenous protein synthesis. MHCC-LM3 cells were seeded at 3×105 cells/well in a 6-well plate and transfected with the indicated constructs. At 48 h after transfection, the cells were treated with 0.1% DMSO (Beijing Solarbio Science & Technology Co., Ltd.), 75 µg/ml CHX (Sigma-Aldrich; Merck KGaA) and 50 µg/ml actinomycin D (Selleck, Inc.) respectively for 6 h. The cells were then lysed for western blotting. The bands were scanned, and the image semi-quantified using ImageJ software (v1.48; National institutes of Health).

Xenograft growth assay

BALB/cA-nu nude male mice (4-week-old) were obtained from the Animal Laboratory of Daping Hospital, Army Medical University. The mice were maintained under standard animal housing conditions (24°C, 60% humidity, 12-h light/dark cycle, free access to food and purified water) and were divided into two groups (n=5 each). A total of 5×106 MHCC-LM3 cells from CSN1 lentivirus-transfected cells and negative control cells were injected subcutaneously into the mice. At 8 days post-injection, tumor appearance was examined in the mice. The subcutaneous tumor size was calculated and recorded every 4 days using a Vernier caliper. The tumor volume (mm3) was calculated as [length × (width)2]/2. At 28 days post-cellular injection, the mice were anesthetized with pentobarbital sodium (100 mg/kg body weight) and sacrificed by cervical dislocation. Subsequently, the tumors were excised, and tumor weights were recorded for analysis. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health. The experimental study design was approved by the Laboratory Animal Welfare and Ethics Committee of the Army Medical University.

Statistical analysis

All statistical analyses were performed using SPSS 20.0 (IBM Corp.) and GraphPad Prism 8.0 (GraphPad Software, Inc.). All results are presented as the mean ± SD of three experiments. Differences between two groups were analyzed using the unpaired Student's t-test. Differences among >2 groups were analyzed using one-way ANOVA followed by Tukey's post hoc test. Wilcoxon signed-rank test (paired data) and Mann-Whitney's U test (unpaired data) were used to analyze the difference in CSN1 expression between HCC and adjacent tissues. Survival analysis was evaluated using the Kaplan-Meier method and the log-rank test. P<0.05 was considered to indicate a statistically significant difference.

Results

Elevated CSN1 expression indicates poor prognosis in patients with HCC

To characterize CSN1 expression in HCC, CSN1 immunohistochemical staining was conducted on a tissue microarray, which included 117 HCC tissue samples and 58 matched adjacent paracancerous tissue samples. Higher levels of CSN1 were observed in HCC tissue, compared with the matched adjacent tissues (Fig. 1A-C). Subsequently, the effects of CSN1 expression on the pathological characteristics and prognosis of patients with HCC were analyzed. CSN1 expression levels correlated with tumor size, lymph node metastasis, vascular invasion, intrahepatic metastasis and tumor stage (Table I). Moreover, the overall survival (17.38 vs. 37.78%; P=0.0285) and the disease-free survival rates (11.30 vs. 25.74%; P=0.0281) of HCC patients with high CSN1 expression were significantly lower compared with patients with low expression (Fig. 1D and E). In addition, CSN1 expression was significantly higher in the HCC cell lines MHCC-LM3, Hep3B and MHCC-97H compared with the normal liver cell line MIHA (Fig. 1F). These results demonstrated that elevated CSN1 levels were associated with poor prognosis.

Table I.

Relationship between the expression of CSN1 and clinicopathological characteristics of patients with hepatocellular carcinoma.

Table I.

Relationship between the expression of CSN1 and clinicopathological characteristics of patients with hepatocellular carcinoma.

Clinicopathological characteristicsCases, n (%)CSN1 expression, mean ± SEMt-valueP-value
Sex
  Male106 (90.6)39.12±2.501.4140.160
  Female11 (9.4)27.52±8.48
Age, years
  ≥5074 (63.3)36.71±3.030.7120.475
  <5043 (36.7)40.29±3.98
HBsAg
  Positive106 (90.6)37.57±2.570.5910.556
  Negative11 (9.4)42.45±6.45
AFP, ng/ml
  ≥40065 (55.6)40.02±3.270.9230.358
  <40052 (44.4)35.54±3.55
Tumor size, cm
  ≥574 (63.2)42.42±2.962.4480.016
  <543 (36.8)30.47±3.90
Differentiation
  High/moderate93 (79.5)37.44±2.710.4760.635
  Low24 (20.5)40.29±5.31
TNM stagea
  I/II64 (54.7)31.21±3.333.2410.002
  III/IV53 (45.3)46.26±3.15
BCLC stageb
  A/B63 (53.8)30.83±3.363.3730.001
  C/D54 (46.2)46.43±3.10
Vascular invasion
  Yes38 (32.5)46.95±3.602.6360.010
  No79 (67.5)33.74±3.01
Intrahepatic metastasis
  Yes40 (34.2)46.25±3.862.5190.013
  No77 (65.8)33.76±2.96
Lymph node metastasis
  Yes12 (10.3)54.00±7.962.2850.024
  No105 (89.7)36.20±2.47

a TNM staging was based on the 8th Edition of The American Joint Committee on Cancer Staging Manual.

b BCLC staging was based on the Edition of the Barcelona-Clinic Liver Cancer Group in 1999. HBsAg, hepatitis B surface antigen; AFP, α-fetoprotein.

CSN1 knockdown inhibits HCC cell proliferation, migration and invasion

To investigate the role of CSN1 in HCC cells, three siRNA candidates were designed and transfected into MHCC-LM3, Hep3B and MHCC-97H cells, and the most efficient siRNA was screened for using western blotting (Fig. 2A). Subsequently, functional analyses were conducted in MHCC-LM3 and Hep3B cells following CSN1 siRNA knockdown. CSN1 knockdown significantly reduced cell proliferation, compared with NC (Fig. 2B). Moreover, cell migration also significantly decreased after CSN1 knockdown (Fig. 2C and D). Lastly, CSN1 knockdown increased the apoptosis rate (Fig. 2E) and affected cell cycle distribution. The proportion of cells in the G0/G1 phase significantly increased, while the proportion of cells in S and G2/M phases significantly decreased after CSN1 knockdown (Fig. 2F). In conclusion, CSN1 knockdown inhibited HCC cell proliferation, migration and invasion.

CSN1 affects HCC cell proliferation and migration via regulating cyclin A2 expression

Cyclin A2 is reported to regulate tumor growth and metastasis (34,35). To investigate the mechanisms by which CSN1 affects HCC proliferation, the expression levels of several cell cycle-related proteins were assessed following CSN1 overexpression. Cyclin A2 and p-CDK4 expressions were upregulated by CSN1 overexpression (Fig. 3A).

Moreover, co-transfection of si-cyclin A2 and OE-CSN1 were carried out. The efficiency of cyclin A2 siRNA transfections was confirmed by western blotting (Fig. 3B). Co-transfection of si-cyclin A2 with OE-CSN1 significantly reduced the effects of OE-CSN1 on cyclin A2 levels, proliferation and migration, compared with cells transduced with OE-CSN1 alone. Thus, cyclin A2 may be a downstream regulator of CSN1 (Fig. 3B-E).

Since CSN subunits may inhibit the ubiquitination and degradation of several protein substrates (22), the effect of CSN1 on cyclin A2 degradation was evaluated. Cyclin A2 mRNA levels were elevated by CSN1 overexpression (Fig. 3F), and the half-life of cyclin A2 protein was not affected by CSN1 overexpression (Fig. 3G). Moreover, inhibition of mRNA transcription or translation by actinomycin D and cycloheximide, respectively, reduced cyclin A2 protein levels in CSN1-overexpressing cells (Fig. 3H), indicating that CSN1 may regulate cyclin A2 expression at the translational level or in an indirect manner. In conclusion, these results indicated that CSN1 promoted HCC cell proliferation and migration by upregulating cyclin A2 expression.

CSN1 facilitates HCC growth in vivo

To further determine the effects of CSN1 on the tumor growth of HCC cells in vivo, a xenograft tumor assay was performed with stably infected OE-CSN1 and NC MHCC-LM3 cells (Fig. 4A). CSN1 overexpression significantly increased the tumor volumes and weights of the MHCC-LM3 cells compared with the negative control group (Fig. 4B). Based on the tumor volumes, CSN1 overexpression was associated with higher tumor growth rate and tumor weights (Fig. 4C and D). These results suggested that CSN1 overexpression can facilitate tumor growth in vivo.

Discussion

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver, accounting for ~85% of all cases (36). The high mortality rate of liver cancer is mainly associated with tumor invasion, metastasis and tumor recurrence after surgical resection (6,37). In recent years, a number of studies have confirmed that the CSN superfamily is widely involved in the regulation of several important intracellular pathways, including cell proliferation, migration, invasion and signal transduction, and plays an important role in tumor development (13,3840). For example, CSN5 and CSN6 are highly expressed in myeloma, lung, colon, breast cancer, malignant glioma and leukemia clinical tissues (14). However, the expression pattern and role of CSN1 in HCC have yet to be determined.

The present study reported that CSN1 expression levels were increased in HCC tissues and HCC cells compared to normal controls. Of note, because of the tumor heterogeneity, 30.5% of the HCC tissues displayed a decrease or no difference in CSN1 expression compared to adjacent tissues. High CSN1 levels indicated poor prognosis in patients. In addition, in vitro assays indicated that CSN1 knockdown inhibited the proliferation and migration of HCC cells, suggesting that CSN1 may promote the occurrence and development of HCC by enhancing proliferation and inhibiting apoptosis. Moreover, the xenograft growth assay revealed that CSN1 facilitated HCC growth in vivo. Mechanically, CSN1 can affect the expression of cyclin A2 and thus participate in the regulation of the cell cycle, cell proliferation and migration. Cyclin A2 is a key regulator of cell cycle transition and can regulate cell growth through its pro-mitotic effects. Cyclin A2 modulates epithelial-mesenchymal transition via β-catenin and phospholipase C pathways (41). The present study supported the notion that CSN1 played a role in carcinogenesis and cancer progression.

The role of the CSN complex in ubiquitin-mediated protein degradation is well-characterized. Several CSN subunits exert de-ubiquitination activity. For example, CSN5 promoted the proliferation of non-small cell lung cancer cells by inhibiting the ubiquitination-mediated degradation of survivin (17). Du et al (42) demonstrated that CSN6 promoted the occurrence of gastric cancer via the ubiquitin-independent proteasomal degradation of p16INK4a. In addition, CSN is also involved in ubiquitin-like modifications. For example, the CSN complex de-neddylates cullins and destabilizes E3 ligase complexes (43). De-neddylation by the CSN complex inactivates a cullin1 complex that ubiquitinates capicua following its phosphorylation by MAP kinase in response to EGFR signaling (44). The CUL4A-RBX1-DDB1-DDB2 complex (CRL4ADDB2) is inactive in the absence of damaged DNA and requires CSN to regulate the repair process (45).

However, the present results indicated that although CSN1 can increase the expression of cyclin A2 mRNA, the regulatory effects of CSN1 on cyclin A2 may not occur at the post-translational level, since the protein half-time of cyclin A2 was not affected by CSN1. There are several possible reasons for this unexpected result. First, CSN1 may not interact with cyclin A2 directly, and there may be other mediators involved in the CSN1-cyclin A2 axis. Secondly, cyclin A2 may not be a direct substrate in CSN-mediated ubiquitination. Moreover, CSN1 might not be the subunit in the CSN complex that mediates the ubiquitination of its substrates. It has been suggested that the MPN domain in CSN6 and CSN5 may be responsible for regulating ubiquitination and cullin de-neddylation (14,46,47). Since CSN1 lacks an MPN domain, there is still no evidence that CSN1 is directly involved in ubiquitination.

There are several limitations in the present study. The exact molecular mechanism of CSN1 in HCC tumorigenesis needs further investigation, especially regarding how CSN1 affects cyclin A2 to regulate HCC cell proliferation and migration. Moreover, how CSN1 and other CSN components interplay to regulate HCC tumorigenesis should also be evaluated in the future.

Taken together, the present findings indicated that high expression levels of CSN1 may contribute to HCC progression. CSN1 knockdown inhibited HCC cell proliferation and induced apoptosis and migration by affecting cyclin A2 expression. The present study determined the function of CSN1 and may provide new insights and therapeutic strategies for HCC prevention and treatment.

Acknowledgements

The authors would like to thank the Hepatobiliary Surgery laboratory of Xinan Hospital, Army Medical University for providing laboratory resources.

Funding

The present study was supported by the National Natural Science Foundation of China (grant no. 81270523).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

PC and JC designed the experiments. HF and YZ performed most of the experiments and drafted the manuscript. YC conducted the experiments on the clinical samples. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Informed consent was obtained from all participants. Experiments involving clinical samples conformed to the principles of the Declaration of Helsinki and were approved by The Ethics Committee of Daping Hospital, Army Medical University. All procedures involving mice and the corresponding experimental protocols were approved by The Laboratory Animal Welfare and Ethics Committee of the Army Medical University.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

AFP

α-fetoprotein

CSN1

constitutive photo morphogenesis 9 signalosome subunit 1

HCC

hepatocellular carcinoma

BCLC

Barcelona Clinic Liver Cancer

HBsAg

hepatitis B surface antigen

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Spandidos Publications style
Fu H, Zhang Y, Chen Y, Chen J and Chen P: CSN1 facilitates proliferation and migration of hepatocellular carcinoma cells by upregulating cyclin A2 expression. Mol Med Rep 23: 46, 2021
APA
Fu, H., Zhang, Y., Chen, Y., Chen, J., & Chen, P. (2021). CSN1 facilitates proliferation and migration of hepatocellular carcinoma cells by upregulating cyclin A2 expression. Molecular Medicine Reports, 23, 46. https://doi.org/10.3892/mmr.2020.11684
MLA
Fu, H., Zhang, Y., Chen, Y., Chen, J., Chen, P."CSN1 facilitates proliferation and migration of hepatocellular carcinoma cells by upregulating cyclin A2 expression". Molecular Medicine Reports 23.1 (2021): 46.
Chicago
Fu, H., Zhang, Y., Chen, Y., Chen, J., Chen, P."CSN1 facilitates proliferation and migration of hepatocellular carcinoma cells by upregulating cyclin A2 expression". Molecular Medicine Reports 23, no. 1 (2021): 46. https://doi.org/10.3892/mmr.2020.11684