Dulbecco's minimum essential medium (DMEM), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), fetal bovine serum (FBS), Lipofectamine® 2000 and Trizol reagent were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). RevertAid First Strand cDNA Synthesis kit was purchased from Fermentas (Thermo Fisher Scientific, Inc.). iQ™ SYBR® Green Supermix was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The following primary rabbit anti-human antibodies were used: PREX2 polyclonal (catalog no., ab121462), phosphorylated (p)-PTEN monoclonal (catalog no., ab109454), PTEN monoclonal (catalog no., ab32199), p-AKT monoclonal (catalog no., ab81283), AKT monoclonal (catalog no., ab32505) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal (catalog no., ab128915) antibodies. All antibodies, including mouse anti-rabbit secondary polyclonal immunoglobulin G horseradish peroxidase antibody (catalog no., ab6728), were purchased from Abcam (Cambridge, MA, USA). An enhanced chemiluminescence (ECL) kit was purchased from Pierce Biotechnology, Inc. (Rockford, IL, USA). PREX2 small interfering (si) RNA was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). PREX2 plasmid was purchased from DNASU Plasmid Repository (Tempe, AZ, USA).

The current study was approved by the Ethics Committee of Yuhuangding Hospital (Yantai, Shandong, China). A total of 15 primary HCC tissues and matched non-tumorous adjacent specimens were collected from 15 patients undergoing liver resection at the Department of Hepatobiliary Surgery at Yuhuangding Hospital. Written informed consent was obtained from each patient. The histomorphology of all the samples were confirmed by the Department of Pathology at Yuhuangding Hospital. HCC tissues were immediately snap-frozen in liquid nitrogen following surgical removal.

Human HCC HepG2, LH86, LMH and PLHC-1 cell lines, and normal human liver THLE-3 cell line were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in DMEM with 10% FBS at 37°C in a humidified incubator containing 5% CO₂.

Total RNA was extracted from the tissue samples using Trizol reagent, according to the manufacturer's protocol. For mRNA detection, RevertAid First Strand cDNA Synthesis kit was used to convert RNA into complementary DNA, according to the manufacturer's protocol. RT-qPCR was performed using iQ SYBR Green Supermix and an Applied Biosystems 7500 thermocycler (Thermo Fisher Scientific, Inc.). The specific primer pairs were as follows: PREX2 sense, 5′-GGACTCCACGGAAACACAGTG-3′ and anti-sense, 5′-GTGAGACTGCCATTCCTCTAAAA-3′; GAPDH, as a reference, sense, 5′-ACAACTTTGGTATCGTGGAAGG-3′ and anti-sense, 5′-GCCATCACGCCACAGTTTC-3′. Independent experiments were repeated three times. The relative expression levels of mRNA were analyzed by use of the 2^−∆∆Cq method.

Lipofectamine 2000 was used to perform cell transfection, according to the manufacturer's protocol. For PREX2 functional analysis, HCC cells were transfected with PREX2 siRNA or pcDNA3.1-PREX2 plasmid.

Tissues and cell lines were solubilized in cold RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). The proteins were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific Inc.). The polyvinylidene difluoride membrane was incubated with phosphate buffered saline containing 5% milk overnight at 4°C. Subsequently, the polyvinylidene difluoride membrane was incubated with Tris-buffered saline and Tween 20 containing 5% milk at room temperature for 3 h, and incubated with rabbit anti-human PREX2 polyclonal (dilution, 1:200) and rabbit anti-human p-PTEN (dilution, 1:500), PTEN (dilution, 1:100), p-AKT, (dilution, 1:100), AKT (dilution, 1:200) and GAPDH (dilution, 1:100) monoclonal antibodies at room temperature for 3 h. Following the incubation, the membrane was incubated again with rabbit anti-mouse secondary antibody (dilution, 1:10,000) at room temperature for 1 h. An ECL kit was used to perform chemiluminescence detection. The relative protein expression was analyzed by Image-Pro® Plus software version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA), represented as the density ratio vs. GAPDH.

MTT assay was used to measure cell proliferation. The cells were cultured in a 96-well plate (10,000 cells/well), each well had 100 µl fresh serum-free medium with 0.5 g/l MTT. Following an incubation at 37°C for 0, 24, 48 and 72 h, the medium was removed by aspiration and 50 µl dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA) was added to each well. Following an incubation at 37°C for 10 min, the A492 of each sample was measured using a plate reader (SM800 Microplate Reader; Shanghai Utrao Medical Instrument Co., Ltd., Shanghai, China).

The cells were collected by incubation with 0.5% trypsin (Thermo Fisher Scientific Inc.) and re-suspended in DMEM containing 10% FBS. Each well of a 24-well plate was seeded with 5×10⁴ cells. When the cells reached ~100% confluence following culturing at 37°C in an atmosphere of 5% CO₂, the bottom of the wells were scratched with a 10 µl pipette tip. The cells were washed with serum-free medium and cultured in DMEM medium containing 10% FBS at 37°C in an atmosphere of 5% CO₂ for 36 h. Images of the cells were captured (DP26 Digital Camera for Microscopy; Olympus Corporation, Tokyo, Japan).

All data are presented as the mean ± standard deviation. One-way analysis of variance were used to analyze the data using SPSS software version 17 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The present study examined the expression of PREX2 in HCC tissues and human HCC HepG2, LH86, LMH and PLHC-1 cell lines. As revealed in Fig. 1A, the expression of PREX2 was increased in HCC tissues compared with the normal matched adjacent tissues. Fig. 1B and C demonstrate that the mRNA and protein expression level of PREX2 was also significantly increased in the HCC cell lines compared with the normal human liver THLE-3 cells. These findings suggest that deregulation of PREX2 is implicated in HCC. Since PLHC-1 cells demonstrated the most significant upregulation of PREX2, the present study used this cell line in subsequent experiments.

The present study investigated the role of PREX2 in the regulation of HCC proliferation in vitro in more detail. The current study transfected PLHC-1 cells with PREX2 plasmid or PREX2 siRNA to modulate the expression level of PREX2. Following transfection, RT-qPCR and western blot analysis were used to determine the mRNA and protein levels of PREX2 in PLHC-1 cells. The results demonstrated that the mRNA and protein levels of PREX2 were upregulated in PLHC-1 cells transfected with PREX2 plasmid, and reduced in PLHC-1 cells transfected with PREX2 siRNA compared with the control cells (control cells, non-transfected PLHC-1 cells; Fig. 2A and B).

Subsequently, an MTT assay was performed to determine cell proliferation in PLHC-1 cells transfected with PREX2 plasmid or PREX2 siRNA. As revealed in Fig. 2C, the proliferation of PLHC-1 cells was significantly reduced following knockdown of PREX2, and the proliferation was notably increased following overexpression of PREX2 compared with the control cells. Therefore, this suggests that PREX2 serves an oncogenic role in the regulation of HCC cell proliferation.

A scratch assay was performed to determine cell migration in PLHC-1 cells transfected with PREX2 plasmid or PREX2 siRNA (Fig. 3A). Following the inhibition of PREX2 expression, the migration of PLHC-1 cells was significantly reduced compared with the control cells (Fig. 3B). Following overexpression of PREX2, the migratory capacity of PLHC-1 cells was notably upregulated compared with the control cells (Fig. 3B). Accordingly, this suggests that PREX2 may serve an oncogenic role in the regulation of HCC cell migration.

PREX2 inhibits the activity of PTEN, and activates AKT signaling (16). Accordingly, the present study investigated the activity of PTEN and AKT signaling pathways in PLHC-1 cells transfected with PREX2 plasmid or PREX2 siRNA by western blot analysis (Fig. 4A). The findings demonstrated that p-PTEN expression levels were increased in PLHC-1 cells transfected with PREX2 plasmid, indicating that the activity of PTEN was reduced. By contrast, the activity of PTEN was upregulated in PLHC-1 cells following knockdown of PREX2. Furthermore, the level of p-AKT was increased in PLHC-1 cells with an overexpression of PREX2, indicating that the activity of the AKT signaling pathway was upregulated (Fig. 4C; P<0.01). By contrast, knockdown of PREX2 downregulated the activity of the AKT signaling pathway in PLHC-1 cells (Fig. 4C; P<0.01). Therefore, these findings indicate that PREX2 inhibits PTEN activity and activates AKT signaling in HCC cells.