In total, 34 pairs of HCC tissues and adjacent normal tissues were obtained from patients (23 males, 11 females; age range 43–72 years; median age, 61 years) who underwent surgical resection at the Affiliated Hospital of Yan'an University (Yan'an, China) between May 2015 and February 2017. Adjacent normal tissues were obtained 2 cm away from HCC tissues. The clinicopathological features of these patients are summarized in Table I. Patients were diagnosed using the tumor-node-metastasis (TNM) system (25). Patients treated with radiotherapy, chemotherapy or other treatments prior to surgery were excluded from the study cohort.

The present study was approved by the Ethics Committee of Affiliated Hospital of Yan'an University. Written informed consent was obtained from all participants.

A total of 2 human HCC Huh7 and Hep3B cell lines and the immortalized normal human liver epithelial L-O2 cell line were purchased from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science (Shanghai, China). All cells were grown at 37°C in a humidified atmosphere containing 5% CO₂ and 95% air, and cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 100 µ/ml penicillin and 100 µg/ml streptomycin (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Cells were plated into 6-well plates with a density of 6×10⁵ cells/well. Then, 12 h after inoculation, cells were transfected with miRNA negative control mimics (miR-NC) or miR-663b mimics (both from Guangzhou Ribobio Co., Ltd., Guangzhou, China) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), as per the manufacturer's protocol. To restore Grb2-associated binding 2 (GAB2) expression, the full length sequence of GAB2 was chemically synthesized by Guangzhou Fueneng Gene Co., Ltd. (Guangzhou, China), cloned into pcDNA3.1 vector and named as pcDNA3.1-GAB2. The transfection efficiencies of miR-663b mimics and pcDNA3.1-GAB2 were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. RT-qPCR and western blot analysis were performed at 48 and 72 h after transfection, and carried out as described in the following paragraphs. Cell Counting kit-8 (CCK-8) and Transwell invasion assays were conducted at 24 and 48 h post-transfection, respectively.

TRIzol^® reagent (Thermo Fisher Scientific, Inc.) was used to isolate total RNA from tissue samples or cells. The concentration of total RNA was measured using a NanoDrop 1000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). For the detection of miR-663b expression, total RNA was subjected to cDNA synthesis using a TaqMan™ MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), and then qPCR was conducted using a TaqMan™ MicroRNA Assay kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The cycling conditions were: 50°C for 2 min, 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 60 sec. To analyze GAB2 mRNA expression, RT was performed to produce cDNA using a PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd., Dalian, China), followed by qPCR with a SYBR Premix Ex Taq™ kit (Takara Biotechnology Co., Ltd.). The thermocycling conditions were: 5 min at 95°C, 40 cycles of 95°C for 30 sec and 65°C for 45 sec. U6 small nuclear RNA and GAPDH were used as internal references for miR-663b and GAB2 expression, respectively. All data was calculated by the 2^−∆∆Cq method (26). The primers were designed as follows: miR-663b, 5′-CGCTAACAGTCTCCAGTC-3′ (forward) and 5′-GCGACACCAAACTGGATGA-3′ (reverse); U6, 5′-CTCGCTTCGGCAGCACATATACT-3′ (forward) and 5′-ACGCTTCACGAATTTGCGTGTC-3′ (reverse); GAB2, 5′-CTGAGACTGATAACGGAGAT-3′ (forward) and 5′-GAGGTGTTTCTGCTTGAC-3′ (reverse); and GAPDH, 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ (forward) and 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′ (reverse).

Transfected cells were incubated at 37°C with 5% CO₂ for 24 h. Next, transfected cells were collected and plated into 96-well plates with a density of 3×10³ cells per well. CCK-8 assays were performed at 0, 1, 2 and 3 days after inoculation to detect cell proliferation, in accordance with the manufacturer's protocol. In detail, 10 µl CCK-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added into each well. Following incubation at 37°C for 2 h, the absorbance was measured at a wavelength of 450 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Transwell plate cell culture chambers (Corning Life Sciences, Cambridge, MA, USA) coated with Matrigel (BD Biosciences, San Jose, CA, USA) were utilized to evaluate invasion capacities of HCC cells. Matrigel coating was performed at 37°C for 2 h. Following transfection for 48 h, cells were harvested, washed and diluted into FBS-free DMEM. A total of 1×10⁵ transfected cells were placed into the upper compartment of the Transwell plate cell culture chambers, and DMEM containing 20% FBS was added into the lower compartments. The chambers were then incubated at 37°C at 5% CO₂ for 24 h. Non-invasive cells remaining on the upper chamber surface were gently removed using a cotton swab. The invasive cells that had passed through the membranes were fixed with 4% paraformaldehyde at room temperature for 20 min and stained with 0.5% crystal violet at room temperature for 20 min. The number of invasive cells was counted in 5 randomly chosen visuals per chamber under an inverted microscope (IX73; magnification, ×200; Olympus Corporation, Tokyo, Japan).

Bioinformatics analysis was applied to predict the putative targets of miR-663b using TargetScan (http://www.targetscan.org/) and microRNA.org (http://www.microrna.org/). GAB2 was predicted as a major target of miR-663b and was considered for subsequent experimental analysis.

The 3′-UTR fragments of GAB2 containing the wild-type (wt) or mutant (mut) miR-663b-binding sequences were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China), inserted into the pGL3 luciferase reporter vector (Promega Corporation, Madison, WI, USA) and named as pGL3-wt-GAB2-3′-UTR and pGL3-mut-GAB2-3′-UTR, respectively. Cells were plated into 24-well plates at a density of 60–70% confluence 12 h prior to transfection. Cells were co-transfected with miR-663b mimics (50 pmol) or miR-NC (50 pmol), and pGL3-wt-GAB2-3′-UTR or pGL3-mut-GAB2-3′-UTR, using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) based on the manufacturer's instructions. Luciferase activity was measured at 48 h post-transfection using the Dual-Luciferase reporter system (Promega Corporation). Renilla luciferase activity was used as an internal reference.

Western blot analysis was performed to detect GAB2 protein expression. Total protein was isolated from homogenized tissues or cultured cells using cold radioimmunoprecipitation assay buffer, and then was subjected to the detection of protein concentration using a BCA Assay kit (both Nanjing KeyGen Biotech Co., Nanjing, Ltd, China). Protein samples (20 µg) were separated on a 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were then blocked at room temperature for 2 h with 5% skimmed milk diluted in TBS/0.1% Tween (TBST) and incubated overnight at 4°C with primary antibodies. Following extensive washing with TBST, the membranes were additionally probed with the goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (cat. no. ab205718; 1:5,000 dilution; Abcam, Cambridge, UK) for 2 h at room temperature, followed by visualization of the protein bands using an enhanced chemiluminescence reagent (Bio-Rad Laboratories, Inc.). The primary antibodies used in the present study included rabbit anti-human GAB2 antibody (cat. no. ab108423; 1:1,000 dilution; Abcam) and rabbit anti-human GAPDH antibody (cat. no. ab128915; 1:1,000 dilution; Abcam). GAPDH was used as a loading control.

All data are presented as the means ± standard deviation, and were analyzed using the SPSS 21.0 software (IBM Corp., Armonk, NY, USA). The differences between two groups were examined using Student's t-test or Wilcoxon signed-rank test, and differences between three or more groups were analyzed using one-way analysis of variance (ANOVA). The Student-Newman-Keuls test was employed as a post-hoc test following ANOVA. The association between miR-663b and GAB2 mRNA levels in HCC tissues was determined using Spearman's correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

To detect the expression status of miR-663b in HCC, miR-663b expression in HCC was first determined by initially comparing the levels of miR-663b in 34 pairs of HCC tissues and adjacent normal tissues. Results from RT-qPCR analysis revealed that the expression levels of miR-663b were decreased in HCC tissues compared with that in adjacent normal tissues (Fig. 1A; P<0.05). miR-663b expression was then additionally measured in 2 human HCC Huh7 and Hep3B cell lines, and an immortalized normal human liver epithelial L-O2 cell line. Significantly decreased expression levels of miR-663b were observed in the 2 HCC cell lines compared with that in L-O2 (Fig. 1B; P<0.05). These results suggest that the decreased miR-663b expression may be associated with the progression and development of HCC.

To examine the biological roles of miR-663b in HCC, Huh7 and Hep3B cells were treated with miR-663 mimics to increase its endogenous expression (Fig. 2A; P<0.05). The effect of miR-663b overexpression on HCC cell proliferation was determined by CCK-8 assay. The results indicated that ectopic miR-663b expression significantly inhibited the proliferation of Huh7 and Hep3B cells (Fig. 2B; P<0.05). In addition, a Transwell invasion assay was applied to detect the invasion ability of Huh7 and Hep3B cells transfected with miR-663b mimics or miR-NC. The invasion ability of Huh7 and Hep3B cells was markedly decreased following treatment with miR-663b mimics (Fig. 2C; P<0.05). Taken together, these results suggested that miR-663b may serve as a tumor suppressor in HCC.

To explore the fundamental mechanisms of miR-663b in HCC cells, bioinformatics analysis was used to predict the potential targets of miR-663b. A total of 135 genes were predicted as candidates of miR-663b, including GAB2, tumor suppressor candidate 2 (TUSC2), sex-determining region Y-box 12, chromodomain-helicase-DNA-binding protein 5, cyclin D2 and transcription factor forkhead box A2. GAB2 was predicted as a major target of miR-663b (Fig. 3A) and was considered for additional experimental analysis as GAB2 has been demonstrated to be closely associated with HCC oncogenesis and development (27–29). To affirm this prediction, a luciferase reporter assay was performed to clarify whether miR-663b may directly interact with the 3′-UTR of GAB2. Fig. 3B indicated that miR-663b overexpression suppressed the luciferase activity of the reporter plasmid with wt 3′-UTR of GAB2 in Huh7 and Hep3B cells (P<0.05); however, the luciferase activity of the reporter plasmid carrying the mut 3′-UTR of GAB2 was unaffected. In order to additionally confirm that GAB2 was a direct target for miR-663b, RT-qPCR and western blot analysis were performed to detect GAB2 mRNA and protein levels in Huh7 and Hep3B cells following transfection with miR-663b or miR-NC. The results demonstrated that mRNA (Fig. 3C; P<0.05) and protein (Fig. 3D; P<0.05) levels of GAB2 in Huh7 and Hep3B cells were markedly downregulated by miR-663b overexpression. Therefore, GAB2 is a direct target of miR-663b in HCC cells.

To additionally investigate the association between miR-663b and GAB2 in HCC, GAB2 expression was measured in 34 pairs of HCC tissues and adjacent normal tissues. The data from the RT-qPCR analysis indicated that GAB2 mRNA levels were upregulated in HCC tissues compared with that in adjacent normal tissues (Fig. 4A; P<0.05). Concomitantly, western blot analysis revealed that the expression level of GAB2 protein was significantly increased in HCC tissues compared with that in adjacent normal tissues (Fig. 4B; P<0.05). Furthermore, Spearman's correlation analysis revealed an inverse correlation between miR-663b and GAB2 mRNA expression in HCC tissues (Fig. 4C; r=−0.5904; P=0.0002). These results suggest that the upregulation of GAB2 in HCC tissues was, at least partly, caused by miR-663b downregulation.

A series of rescue experiments were performed to determine whether GAB2 overexpression may reverse cell proliferation and invasion suppression caused by miR-663b upregulation in Huh7 and Hep3B cells. Therefore, pcDNA3.1-GAB2 or empty pcDNA3.1 plasmids were transfected into Huh7 and Hep3B cells, and western blot analysis was performed to detect GAB2 protein expression. GAB2 protein levels were significantly increased in pcDNA3.1-GAB2-transfected Huh7 and Hep3B cells compared with that in cells transfected with pcDNA3.1 (Fig. 5A; P<0.05). Western blot analysis also identified that the downregulation of GAB2 protein induced by miR-663b overexpression was restored in Huh7 and Hep3B cells by co-transfecting with pcDNA3.1-GAB2 (Fig. 5B and C; P<0.05). Furthermore, CCK-8 and Transwell invasion assays demonstrated that GAB2 overexpression partially rescued the inhibitory effects of miR-663b on the proliferation (Fig. 5D; P<0.05) and invasion (Fig. 5E; P<0.05) of Huh7 and Hep3B cells. These data confirmed that miR-663b may serve inhibitory roles in HCC, at least partly, by decreasing GAB2 expression.