Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), Lipofectamine 2000, TRIzol reagent and the miRNA Reverse Transcription kit were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). miRNA quantitative polymerase chain reaction (qPCR) Detection kit was purchased from Genecopoeia (Rockville, MD, USA). Directed Mutagenesis kit was purchased from Agilent Technologies, Inc. (Santa Clara, CA, USA). Rabbit anti-human Spry2 monoclonal antibody (cat. no. ab180527), rabbit anti-human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (cat. no. ab8245) and mouse anti-rabbit secondary antibody IgG (HRP; cat. no. ab6728) were purchased from Abcam (Cambridge, MA, USA). Enhanced chemiluminescence (ECL) kit was purchased from Pierce Protein Biology; Thermo Fisher Scientific, Inc. (Rockford, IL, USA). PsiCHECK 2 vector was purchased from Promega Corporation (Madison, WI, USA). miR-27b mimics, miR-27b inhibitor, Spry2 plasmid and Spry2-specific small interfering (si)RNA were obtained from Guangzhou FulenGen Co., Ltd. (Guangzhou, China).

The present study was approved by the Ethics Committee of Yuhuangding Hospital (Yantai, China). A total of 35 primary HCC tissues and matched normal adjacent specimens were collected. Written informed consent was obtained. The histomorphology of all samples was confirmed by the Department of Pathology. HCC tissues were immediately snap-frozen in liquid nitrogen upon surgical removal.

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

Total RNA was extracted from tissues and cells using TRIzol reagent. Tissues were homogenized prior to RNA extraction by grinding. All samples were treated with 1 µl DNase (Thermo Fisher Scientific, Inc.) prior to RT. The miRNA Reverse Transcription kit was used to reverse transcribe RNA into cDNA, according to the manufacturer's protocol. Briefly, reverse transcription was performed at 16°C for 30 min, followed by an incubation step at 42°C for 30 min and enzyme inactivation at 85°C for 5 min. qPCR was performed using a miRNA qPCR Detection kit on an ABI 7500 thermocycler (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The primers for Spry2 were: Forward: 5′-CCTACTGTCGTCCCAAGACCT-3′ and reverse: 5′-GGGGCTCGTGCAGAAGAAT-3′ purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Briefly, the PCR cycling conditions were as follows: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing/elongation at 60°C for 60 sec. U6 was used as an internal reference gene. The experiment was repeated 3 times. The relative expression levels were analyzed using the 2^−ΔΔCq method (15).

Tissues and cells were solubilized in cold radioimmunoprecipitation lysis buffer (Beyotime Biotechnology, Shanghai, China). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (50 µg of protein/lane), and transferred onto a polyvinylidene difluoride (PVDF; Thermo Fisher Scientific, Inc.) membrane. The membrane was incubated with phosphate-buffered saline containing 5% milk overnight at 4°C. Subsequently, the PVDF membrane was incubated with rabbit anti-Spry2 monoclonal antibody (1:50) and rabbit anti-GAPDH monoclonal antibody (1:50) at room temperature for 3 h. The membrane was then incubated with mouse anti-rabbit secondary antibody (1:5,000) at room temperature for 1 h. An ECL kit was used to perform chemiluminescent detection. The protein concentration was quantified using Pierce BCA Protein Assay Kit, Thermo Fisher Scientific, Inc. The relative protein expression levels were analyzed using Image-Pro Plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA), and are presented as the density ratio vs. GAPDH.

Lipofectamine 2000 was used to transfect the HepG2 cells, according to the manufacturer's protocol. Briefly, cells were cultured to 70% confluence, at a seed density of 10⁶ and resuspended in serum-free medium. miR-27b mimics, miR-27b inhibitor, Spry2 plasmid (Syngentech, Beijing, China), Spry2-specific siRNA and Lipofectamine 2000 were diluted with serum-free medium. The diluted Lipofectamine 2000 was added to the diluted siRNA and miR-27b mimics, and the mixture was incubated for 20 min at room temperature. Subsequently, the mixture was added to the cell suspension. After a 6 h incubation at 37°C and 5% CO₂, the medium was replaced with the normal serum-containing medium. Subsequently, the cells were cultured for 24 h prior to further experimentation.

The miR-27b-predicted target sequence within the Spry2 3′-UTR (UUUGCAACUGUGAA), and a mutant lacking complimentarity with the miR-27b seed sequence (UUUGCACACACACC), were cloned downstream of the luciferase gene driven by the cytomegalovirus promoter, thus generating Luc.Spry2 and Luc.mtSpry2 vectors, respectively. The wild type (WT) and mutant (MUT) 3′UTRs of Spry2 were obtained from Biowit Technologies (Shenzhen, China), which was inserted into the BgIII and KpnI restriction sites 3′ to the end of the Renilla luciferase gene in the PsiCHECK vector. HepG2 cells were transfected with Luc.Spry2 or Luc.mtSpry2 vectors in conjunction with miR-27b mimics or scramble negative control miR mimics using Lipofectamine 2000. A total of 24 h post-transfection, the cells were harvested. Dual-Luciferase Reporter Assay System (Promega Corporation) was used to determine luciferase activity. Luciferase activity was quantified using an Lmax multiwell luminometer (Molecular Devices, Sunnyvale, CA, USA).

Wound healing assay was performed, in order to evaluate the cell migratory capacity of each group. Briefly, cells were cultured to full confluence. Wounds ~1 mm wide were created in the cell layer using a plastic scriber, and cells were washed with phosphate-buffered saline and incubated in a serum-free medium. A total of 24 h after the wound was generated, cells were incubated in DMEM supplemented with 10% FBS. After further incubation for 0 and 48 h, the cells were fixed with absolute alcohol and observed under a BX53 fluorescence microscope (Olympus Corporation, Tokyo, Japan).

Transwell assay was performed in order to examine cell invasion. Briefly, the invasive ability of HepG2 cells was determined in 24-well Transwell chambers, which were coated with a layer of Matrigel. Cell suspension of DMEM (10⁶ cells/ml) was added to the upper chamber, and DMEM supplemented with 10% FBS was added to the lower chamber. Following a 24 h incubation, non-invading cells as well as the Matrigel (Thermo Fisher Scientific, Inc.) on the interior of the inserts was removed using a cotton-tipped swab. Invasive cells on the lower surface of the membrane were stained with gentian violet, rinsed with water and air-dried. Five fields were randomly selected and cell number was counted under a microscope.

The target genes of miR-27 were predicted using TargetScan (16). The following settings were used for analysis: i) Species, human; ii) gene symbol, Spry2 and iii) selelcted a conserved miRNA family.

Data are presented as the mean ± standard deviation. Statistical analysis was performed using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). The association between miR-27b expression and clinicopathologic features in patients with HCC was analyzed using χ² test and Spearman rank correlation analysis. The statistical correlation of data between the cell groups was analyzed by one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of miR-27b were examined in four common human HCC cell lines, HepG2, LH86, LMH and PLHC-1, as well as in a normal liver cell line, THLE-3. HepG2 cells exhibited a significant increase in the expression levels of miR-27b, as compared with the normal liver THLE-3 cells (Fig. 1A), therefore this cell line was used in further experiments. In addition, the expression levels of miR-27b were significantly increased in the HCC tissues, as compared with in the peritumoral noncancerous tissues (Fig. 1B). The expression levels of miR-27b were increased in 77.1% (27/35) of the HCC tissue samples.

The association between miR-27b expression and clinicopathologic features in patients with HCC was analyzed using χ² test. As presented in Table I, miR-27b expression had no association with the age, gender and tumor size of the patients (P>0.05). However, miR-27b expression was correlated with tumor differentiation, Tumor Node Metastasis (TNM) stage and vascular invasion (P<0.05).

The present study investigated the effects of miR-27b on the regulation of migration and invasion in HCC cells. Since miR-27b was upregulated in HCC cells, HepG2 cells were transfected with a miR-27b inhibitor to suppress its expression. Post-transfection, the expression levels of miR-27b were reduced (Fig. 2A). Wound healing and Transwell assays were conducted to investigate the effects of miR-27b knock-down on HCC cell migration and invasion, respectively. As shown in Fig. 2B and C, knockdown of miR-27b expression significantly inhibited the migration and invasion of HepG2 cells. These results indicate that miR-27b has a role in the regulation of HCC metastasis.

Bioinformatical analysis was performed in order to predict the target genes of miR-27b, and Spry2 was identified as a putative target. To clarify whether Spry2 is a direct target of miR-27b, a Luc.Spry2 vector containing the wild-type 3′-UTR of Spry2 and a Luc.mtSpry2 vector containing a mutated form of the 3′-UTR of Spry2 were generated (Fig. 3A). Subsequently, HepG2 cells were transfected with Luc.Spry2 or Luc.mtSpry2 vector, with or without miR-27b mimics. A total of 24 h post-transfection, the luciferase activity was examined. Compared with the control group, luciferase activity was significantly decreased in the HepG2 cells co-transfected with the Luc.Spry2 vector and miR-27b mimics, but was unaltered in HepG2 cells co-transfected with the Luc.mtSpry2 vector and miR-27b mimics (Fig. 3B). These results indicate that miR-27b directly binds to the seed sequences within the Spry2 3′-UTR in HepG2 cells.

The present study investigated the effects of miR-27b overexpression or knockdown on the protein expression levels of Spry2 in HepG2 cells. HepG2 cells were transfected with miR-27b mimics or a miR-27b inhibitor. As shown in Fig. 3C, transfection with miR-27b mimics upregulated the miR-27b expression levels; however, transfection with the miR-27b inhibitor downregulated miR-27b expression levels. Subsequently, western blotting was performed to examine the protein expression levels of Spry2. As shown in Fig. 3D, upregulation of miR-27b significantly inhibited the protein expression levels of Spry2; however, knockdown of miR-27b expression enhanced the protein expression levels of Spry2 in HepG2 cells. Accordingly, these results suggest that miR-27b may negatively regulate the protein expression levels of Spry2 in HCC HepG2 cells via directly targeting its mRNA.

Since knockdown of miR-27b upregulated Spry2 expression and suppressed HCC cell migration and invasion, the present study hypothesized that Spry2 may be involved in miR-27b-mediated migration and invasion of HCC cells. To verify this hypothesis, HepG2 cells were transfected with a Spry2 plasmid. Post-transfection, the protein expression levels of Spry2 were significantly increased (Fig. 4A). Consistent with miR-27b knockdown, overexpression of Spry2 suppressed HCC cell migration and invasion (Fig. 4B and C).

To further clarify whether the effects of miR-27b on HCC cell migration and invasion were mediated by inhibition of Spry2 expression, HepG2 cells were transfected with a miR-27b inhibitor, or co-transfected with a miR-27b inhibitor and Spry2 siRNA. Post-transfection, the protein expression levels of Spry2 were reduced in HepG2 cells co-transfected with a miR-27b inhibitor and Spry2 siRNA, as compared with the HepG2 cells transfected with a miR-27b inhibitor only (Fig. 5A). In addition, the migratory and invasive capacity was increased in HepG2 cells co-transfected with a miR-27b inhibitor and Spry2 siRNA, as compared with the HepG2 cells transfected with a miR-27b inhibitor only (Fig. 5B and C). These results indicate that knockdown of Spry2 expression may reverse the suppressive effects of miR-27b inhibition on HCC cell migration and invasion. Based on these findings, miR-27b may have a promoting role in the regulation of HCC cell migration and invasion, at least partially via directly targeting Spry2 expression.