For the use of tissue samples, written informed consent was obtained from all patients involved in the present study. The present study was approved by the Medical Ethics Committee of The Second Hospital of Hebei Medical University (Shijiazhuang, China). A total of 42 pairs of HCC tissues and corresponding adjacent non-tumor tissues were collected from patients with HCC (24 males, 18 females) who had undergone surgery treatment at The Second Hospital of Hebei Medical University. None of these patients received chemotherapy or radiotherapy prior to surgery. Tissues were put into liquid nitrogen immediately following surgery and then stored at −80°C until use.

The human HCC HepG2 and SMMC-7721 cell lines and immortalized normal liver epithelial THLE-3 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). All cell lines were maintained under the conditions stated by the supplier.

Mature miR-592 mimics, miR mimics negative control (NC) and luciferase reporter vector were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). Prior to cell transfection for 1 h, cell culture medium was replaced with Dulbecco's modified Eagle's medium (DMEM) medium without antibiotics and fetal bovine serum (FBS) (both from Gibco; Thermo Fisher Scientific, Inc. Waltham, MA, USA). Cells were transfected with miR-592 mimics, NC, or co-transfected with the luciferase reporter vector using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Subsequent to transfection for 4–6 h, cells were washed with PBS and cultured at 37°C with 5% CO₂ according to the manufacturer's conditions in DMEM without antibiotics.

TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from tissues and cells, according to the manufacturer's protocol. The concentration and purity of total RNA was measured by ND-2000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). cDNA was then synthesized using TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) with 1 µg RNA. Subsequent to RT, qPCR was performed using TaqMan MicroRNA assay kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RT-qPCR reaction was performed using an Applied Biosystems 7500 Real-time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The RT-qPCR was performed as follows: 40 cycles of denaturation at 95°C (15 sec) and annealing/extension at 60°C (60 sec). Each sample was analyzed in triplicate, and U6 was used as an internal control. The primer sequences for miR-592 were as follows: Forward, 5′-CCATGACATTGTGTCAATATGCGA-3′ and reverse, 5′-CGTCATGATGTTGCGTCACC-3′. For U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. Relative expression of miR-592 was analyzed using the 2^−ΔΔCq method (20).

Cellular viability was assessed using an MTT assay (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany). Following a 24-h transfection, transfected cells (miR-592 and NC) were seeded in 96-well plates at a density of 2,500 cells/well. Subsequent to being incubated at 37°C for 24, 48, 72 and 96 h, 5 µl MTT solution (5 mg/ml) was added into each well and incubated for 4 h at 37°C. Cell culture medium was removed carefully and replaced with 200 µl dimethyl sulfoxide (Sigma-Aldrich; Merck Millipore). Absorbance at 490 nm was detected using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All experiments (5 replicates in each) were repeated ≥3 times.

The cellular migration assay was assessed using Transwell chambers with a pore size of 8 µm (Corning Incorporated, Cambridge, MA, USA). Following transfection for 48 h, 5×10⁴ transfected cells (miR-592 and NC) in 300 µl medium without FBS were seeded into the upper chamber of the Transwell, while 500 µl medium supplemented with 20% FBS was placed into the lower chamber. For the Matrigel invasion assay, the Transwell chamber was coated with Matrigel (BD Biosciences, San Jose, CA, USA). A total of 5×10⁴ transfected cells (miR-592 and NC) in 300 µl medium without FBS were seeded into the upper chamber of the Transwell, while 500 µl medium supplemented with 20% FBS was placed into the lower chamber. Cells were incubated at 37°C for a further 24 h for the migration assay and 48 h for the invasion assay. In the two assays, the cells were fixed with 100% methanol for 5 min (Beyotime Institute of Biotechnology, Haimen, China) and stained with 0.5% crystal violet (Beyotime Institute of Biotechnology) for 5 min. Following this, cells remaining on the upper surface of the membranes were removed carefully using cotton swabs. The migrated and invaded cells were then counted in five randomly selected fields with an inverted microscope (Olympus Corporation, Tokyo, Japan). Each experiment was repeated ≥times.

The potential target genes of miR-592 were generated using publicly available databases: miRanda (Memorial Sloan-Kettering Cancer Center, New York, NY, USA) and TargetScan (Whitehead Institute for Biomedical Research, Cambridge, MA, USA).

Western blot analysis was performed according to the standard protocol. Following 72-h transfection, transfected cells (miR-592 and NC) were washed and harvested using radioimmunoprecipitation assay lysis buffer (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, along with a protease inhibitor (Pierce; Thermo Fisher Scientific, Inc.). Total protein concentration was measured using a bicinchoninic assay protein assay kit (Beyotime Institute of Biotechnology). Equal amounts of protein (20 µg) were then separated by 10% SDS-PAGE (Beyotime Institute of Biotechnology) and transferred to polyvinylidene difluoride membranes (Merck Millipore). Following a blocking incubation with 5% non-fat milk at room temperature for 2 h, the membranes were incubated at 4°C overnight with primary anti-IGF-1R (dilution, 1:1,000; cat no. ab131476; Abcam, Cambridge, MA, USA) and anti-GAPDH (dilution, 1:1,000; cat no. ab201822; Abcam), followed by incubation at room temperature for 1 h with the goat anti-rabbit horseradish peroxidase conjugated secondary antibody (1:3,000 dilution; cat no. ab97051; Abcam). The proteins were detected with an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) and visualized using the FluorChem imaging system (Alpha Innotech, San Leandro, CA, USA). GAPDH was used as an internal control.

The dual-luciferase report assay was performed to determine whether miR-592 directly targeted the 3′UTR of IGF-1R. Cells were seeded in 24-well plates at a density of 50–60% confluence, and were co-transfected with 100 ng PGL3-IGF-1R-3′UTR wild type (Wt) or PGL3-IGF-1R-3′UTR mutant (Mut), and 100 nM miR-592 mimic or NC, using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Following a 48-h transfection, the firefly luciferase activity was detected using the dual-luciferase reporter assay (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. The Renilla luciferase activity was used as an internal control. Each experiment (3 replicates in each) was repeated >3 times.

Data are presented as the mean ± standard deviation. A two-tailed Student's t-test or ANOVA was used to compare differences between groups using SPSS 19.0 (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

The present study detected the expression of miR-592 in HCC tissue samples and the corresponding adjacent non-tumor tissues using RT-qPCR. It was observed that miR-592 was significantly downregulated in HCC tissues compared with in corresponding adjacent non-tumor tissues (Fig. 1A and B; P=0.019).

The expression of miR-592 in HCC HepG2 and SMMC-7721 cell lines and immortalized normal liver epithelial THLE-3 cells was also measured. As presented in Fig. 1C, miR-592 expression levels were also decreased in HepG2 (P=0.011) and SMMC-7721 (P=0.015) cells compared with THLE-3. Therefore, miR-592 may perform important functions in HCC carcinogenesis and progression.

Statistical analysis was also performed to assess the association of miR-592 expression with clinicopathological factors in patients with HCC. As presented in Table I, low miR-592 expression was significantly associated with TNM stage (P=0.010) and lymph node metastasis (P=0.001). However, no correlation was observed between miR-592 expression and other clinicopathological factors, including age (P=0.750), gender (P=0.754) and differentiation (P=0.330).

To clarify the functions of miR-592 on HCC, miR-592 mimics were transfected into HepG2 and SMMC-7721 cells. The present study analyzed the expression of miR-592 subsequent to a 48-h transfection using RT-qPCR. As demonstrated in Fig. 2, miR-592 was significantly upregulated in HCC HepG2 (P<0.001) and SMMC-7721 (P<0.001) cells transfected with miR-592 mimics, compared with cells transfected with NC.

To explore the functional roles of miR-592, the present study additionally investigated the function of miR-592 on HCC cellular proliferation by using an MTT assay. As presented in Fig. 3, miR-592 significantly decreased cellular proliferation at 72 h and 96 h in HCC HepG2 (P=0.030) and SMMC-7721 (P=0.023) cells. These results suggest that the aberrant expression of miR-592 could regulate HCC cell growth.

A cellular migration and invasion assay was performed to assess the role of miR-592 on HCC cellular motility. As demonstrated in Fig. 4A, miR-592 decreased HCC HepG2 (P=0.034) and SMMC-7721 (P=0.027) cellular migration ability. miR-592 also inhibited the HCC HepG2 (P=0.021) and SMMC-7721 (P=0.015) cellular invasion ability, compared with cells transfected with NC (Fig. 4B). These results indicate that the abnormal expression of miR-592 may be capable of regulating HCC metastasis.

As miR-592 contributed to HCC carcinogenesis and progression, the present study aimed to identify the potential mechanism by which miR-592 could regulate HCC cellular growth, migration and invasion. To investigate the target mRNA of miR-592, the bioinformatics software miRanda and TargetScan were used. As presented in Fig. 5A, the bioinformatics software predicated that IGF-1R mRNA contained a miR-592 seed match at position 1,745–1,752 of the IGF-1R 3′UTR.

To explore the effect of miR-592 on its target IGF-1R protein expression level, western blot analysis was performed. As presented in Fig. 5B, IGF-1R was significantly downregulated in HCC HepG2 (P=0.018) and SMMC-7721 (P=0.013) cells transfected with miR-592.

A dual luciferase reporter assay was performed to verify whether IGF-1R was a direct target of miR-592. As presented in Fig. 6, miR-592 significantly inhibited the luciferase activity following co-transfection with miR-592 mimics and the PGL3-IGF-1R-3′UTR Wt (P=0.025 for HepG2; P=0.014 for SMMC-7721), whereas luciferase activity was no different when miR-592 mimics were co-transfected with PGL3-IGF-1R-3′UTR Mut. Notably, IGF-1R was a direct target of miR-592 in vitro.