A total of eight pairs of HCC tissues and their adjacent normal tissues were obtained from patients who had undergone surgery at the Department of General Surgery, Qianfoshan Hospital, Shandong University (Jinan, China). The present study was approved by the Hospital Institutional Review Board of Qianfoshan Hospital, Shandong University. Written informed consent was obtained from the patient’s family. The HepG2, SMMC-7721 and BEL-7402 cell lines were obtained from Shandong Province Key Laboratory of General Surgery Center (Jinan, China). The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (Invitrogen Life Technologies, Carlsbad, CA, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma, St Louis, MO, USA) at 37°C in 5% CO₂.

The BEL-7402 cells were transfected with either pcDNA or pcDNA-Cat B using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer’s instructions. The HepG2 and SMMC-7721 cells were transfected with either 40 nM Cat B or control siRNA (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 80% confluence using Geneporter 2 transfection reagent (Genlantis, San Diego, CA, USA) according to the manufacturer’s instructions.

Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. A SYBR RT-PCR kit (Takara Bio, Inc., Shiga, Japan) was used for RT-qPCR analysis. The following specific primers were used for RT-qPCR assays: Cat B, forward 5′-CCAGG GAGCA AGACAGAGAC-3′ and reverse 5′-GAGAC TGGCG TTCTC CAAAG-3′ and β-actin, forward 5′-GGCCT CCAAG GAGTA AGACC-3′ and reverse 5′-AGGGG TCTAC ATGGC AACTG-3′. Data from each sample were normalized to the expression of β-actin.

Male, 4-week-old BALB/c nude mice were purchased from the Shanghai Laboratory Animal Company (Shanghai, China). HepG2 cells (1×10⁵) with stable knock down of Cat B and the negative controls were injected subcutaneously under the front legs of the nude mice. The mice were observed over 5 weeks for tumor formation. Subsequently, the mice were sacrificed and, following tumor removal, the wet weights of each tumor were determined.

To prepare the proteins, the treated cell monolayers were rinsed twice using cold phosphate-buffered saline (PBS) and then scraped and transferred into cell lysis buffer containing 50 mM Tris-HCL (pH 7.5), 150 mM NaCl and 0.5% Nonidet P40 (Sigma) with Roche complete EDTA-free protease inhibitor cocktail (Sigma). These suspensions were then centrifuged using a Beckman centrifuge at 10,000 × g for 10 min and the protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal quantities of the proteins were separated using SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) for western blot analysis using primary antibodies against human Cat B (Abcam, Cambridge, UK), phosphorylated Akt (Ser473), Akt (Cell Signaling Technology, Inc., Danvers, MA, USA), β-actin and secondary polyclonal goat anti-rabbit antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Inc.). Following washing with tris-buffered saline with Tween 20 (Beyotime Institute of Biotechnology, Haimen, China), bound antibodies were detected using enhanced chemiluminescence (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Cell viability was assessed using a Cell counting kit-8 (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions.

An annexin-V assay was performed using an Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit according to the manufacturer’s instructions (BD Biosciences, San Diego, CA, USA). Briefly, 2×10⁵ cells were collected, washed twice with cold PBS, resuspended in 100 μl binding buffer containing Hepes (10 mM), NaOH (pH 7.4), NaCl (140 mM) and CaCl₂ (2.5 mM) and incubated with Annexin V-FITC at room temperature for 10 min. This was followed by the addition of 6 μl propidium iodide (PI; 20 μg/ml) for an additional 5 mins. The fluorescent intensities were determined using flow cytometry (Beckman Coulter, Miami, FL, USA).

All data are expressed as the mean ± standard deviation of three or four experiments. Analysis was performed using Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

The expression of Cat B was compared between the HCC tissues and the adjacent normal tissues using RT-qPCR analysis. The results demonstrated that the expression of Cat B was markedly increased in cancer tissues (Fig. 1A). The upregulation of Cat B was also confirmed by representative western blot analysis using the proteins extracted from the tissues (Fig. 1B). Furthermore, several HCC cell lines were selected to detect the expression of Cat B and the results revealed that the expression of Cat B was highest in the HepG2 cells and lowest in the BEL-7402 cells (Fig. 1C). Taken together, these data suggested that Cat B was overexpressed in the HCC tissues and cell lines.

To determine the effect of Cat B on HCC cell growth, the BEL-7402 cells were transfected with either the pcDNA3-Cat B or pcDNA3-vector (mock; Fig. 2A). Cell growth was significantly increased in the Cat B-overexpressed cells compared with their corresponding controls (Fig. 2B). In addition, the cells overexpressing Cat B had a high rate of proliferation (Fig. 2C) and the Annexin V/PI analysis of the Cat B-overexpressed cells revealed a significant decrease in the rate of apoptosis (Fig. 2D).

The upregulation of Cat B observed in the present study prompted further investigation to determine the effect of its knock down on HCC cell growth. The HepG2 cells were infected with either a lentivirus targeting Cat B (shRNA-Cat B) or a negative control (shRNA-Ctrl). As shown in Fig. 3A–B, endogenous Cat B was significantly downregulated by shRNA-Cat B. Consequently, cell growth was significantly decreased in the cells transfected with shRNA-Cat B compared with shRNA-Con (Fig. 3C). These cells also had a lower rate of proliferation (Fig. 3D).

To further determine the role of Cat B knockdown, HepG2 cells with stable knock down of Cat B or negative controls were injected subcutaneously under the front legs of the nude mice and tumor growth was closely monitored for 4 weeks. The results demonstrated that the tumor size and weight were markedly reduced following Cat B knockdown compared with the control (Fig. 4A–B), suggesting that Cat B inhibition suppresses HCC growth in vivo.

It has been demonstrated that Cat B promotes tumor progression, not only by proteolytic function, but also by a series of signal transduction pathways (10,11). Modulation of the PI3K signaling pathway is important in tumor growth. Therefore, in the present study, this intracellular signaling pathway was analyzed in HCC cells by manipulating the levels of Cat B. As shown in Fig. 5A and B, the total level of Akt level was unaltered despite the varying levels of Cat B. However, the phosphorylation of Akt was markedly increased in the cells overexpressing Cat B compared with the control vector-transfected cells (Fig. 5A). By contrast, the phosphorylation of Akt was markedly decreased in the shRNA-Cat B cells compared with the shRNA-control cells (Fig. 5B).

Our previous study demonstrated that the expression of integrin αvβ3 was higher than normal in primary liver cancer and that antisense integrin αvβ3 suppresses the growth of subcutaneously implanted HCC by inhibiting tumor angiogenesis (12). Additionally, it has been suggested that the interaction between integrins and extracellular components is important in tumor differentiation and progression (13). Therefore, the present study hypothesized that Cat B was involved in the progression of hepatoma through integrin αvβ3 to ultimately affect the phosphorylation of Akt. To verify this hypothesis, BEL-7402 cells overexpressing pcDNA3-Cat B were pretreated with either an integrin αvβ3 inhibitory antibody or with an isotype-matched control, immunoglobulin G1. The main components of the PI3K signaling pathway were then detected by western blot analysis. Inhibiting integrin αvβ3 significantly prevented the Cat B-overexpressing phosphorylation of Akt (Fig. 5C), limited Cat B-induced proliferation (Fig. 5D) and led to the apoptosis of Cat B-overexpressing cells (Fig. 5E). Taken together, these results demonstrated that Cat B regulated HCC growth by integrin αvβ3 and subsequently by the PI3K/Akt signaling pathway.