All patients and their families agreed to participate in the present study and gave written informed consent. The present study was approved by the institutional ethics board of The First Affiliated Hospital of Chongqing Medical University (Chongqing, China) and complied with the Declaration of Helsinki.

The human HCC cell lines SMMC-7721, Hep3B and Bel-7402, in addition to normal human hepatocyte HL-7702 cells, were purchased from the China Center for Type Culture Collection (Wuhan, China). Hep3B cells were cultured in Minimum Essential Medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and the other cells were cultured in RPMI-1640 medium (1X; Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; PAN-Biotech GmbH, Aidenbach, Germany) at 37°C in a humidified chamber supplemented with 5% CO₂.

A total of 45 frozen primary tumor samples and corresponding non-cancerous samples (located >3 cm away from the tumor) were obtained from patients undergoing hepatectomy at The First Affiliated Hospital of Chongqing Medical University between September 2015 and March 2016. Patients (9 female and 36 male) were aged between 22 and 78 years and were at the following tumor stages: 4 cases of stage I; 10 cases of stage II; and 31 cases of stage III. Tissue samples were snap frozen in liquid nitrogen at the time of surgery and stored at −80°C. The tumor, node, metastasis (TNM) classification of the Union for International Cancer Control was used. None of the patients received radiotherapy or chemotherapy prior to surgery.

miRNA and mRNA was extracted from cell lines or tissue using Hipure Universal miRNA kit (Magen, Guangzhou, China; http://www.magentec.com.cn), according to the manufacturer's protocol. The primers for miRNA and mRNA were produced using All-in-One™ miRNA qPCR Primer and All-in-One™ mRNA qPCR Primer (GeneCopoeia, Inc., Rockville, MD, USA), and the sequences were: miR-340 forward, 5-'GCG GTT ATA AAG CAA TGA GA-3′ and reverse, 5′-GTGCGTGTCGTGGAGTCG-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; SKP2 forward, 5′-AGTCTCTATGGCAGACCTTAGACC-3′ and reverse, 5′-TTTCTGGAGATTCTTTCTGTAGCC-3′; and GAPDH forward, 5′-CAGTCAGCCGCATCTTCTTTT-3′ and reverse, 5′-GTGACCAGGCGCCCAATAC-3′. The synthesis of cDNA from miRNA and mRNA used All-in-One™ miRNA First-Strand cDNA Synthesis and All-in-One™ First-Strand cDNA Synthesis kits (GeneCopoeia, Inc.). The reaction conditions for miRNA were as follows: 37°C for 60 min; 85°C for 5 min; 4°C holding, and the reaction system had a volume of 20 µl. The reaction conditions for mRNA were as follows: 60°C for 5 min, with a reaction system of 13 µl; 37°C for 60 min; 85°C for 5 min; 4°C holding, and the reaction system had a volume of 25 µl. All steps were performed according to the manufacturer's protocol. qPCR was performed in triplicate for each case using All-in-One™ miRNA kit and All-in-One™ qPCR Mix (GeneCopoeia, Inc.), respectively, on a CFX96™ Real-Time System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The temperature protocol for the reaction was as follows: 95°C for 10 min; 40 cycles at 95°C for 10 sec and 60°C for 20 sec, and 72°C for 15 sec. miRNA expression was measured using Cq (threshold cycle). The 2^−ΔΔCq method for relative quantitation of gene expression was used to determine miRNA expression levels (17). The ΔCq was calculated by subtracting the Cq of U6 RNA from the Cq of the miRNA of interest. Fold changes were generated using the equation 2^−ΔΔCq. The expression level of U6 and GAPDH was used as the internal control for miRNA and mRNA expression, respectively.

The miR-340 mimics (pLVX-ZsGreen-Puro-miR-340), miR-340 inhibitor (pLVX-tdTomato-Puro-miR-340 inhibitor) and their controls (pLVX-ZsGreen and pLVX-tdTomato) were synthesized by GeneCopoeia, Inc. and transfected into the cells at a final oligonucleotide concentration of 1 µg/ml. All cell transfections were performed using EndoFection™-Max (GeneCopoeia, Inc.), according to the manufacturer's protocol. The density of cells was 3×10⁷ per 6-well plate and subsequent experimentation was performed 48 h after transfected.

For the analysis of cell proliferation, cells were seeded into 96-well plates at 1×10⁵ cells/well. Cells were incubated in 10% Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) solution and × diluted in normal culture medium at 37°C until visual color conversion was apparent. The SMMC-7721 proliferation rate was determined at 24, 48 and 72 h following transfection. The absorbance in each well was measured with a VARIOSKAN FLASH (Thermo Fisher Scientific, Inc.) at 450 nm. For the analysis of cellular apoptosis, cells were transfected with miR-340 mimic, miR-340 inhibitor and their corresponding control for 24 h. Following transfection, cells were harvested and washed twice in PBS; miR-340 mimic and its control were stained with an Annexin V-phycoerythrin/7-aminoactinomycin D apoptosis kit [Multi Sciences (Lianke) Biotech. Co., Ltd., Hangzhou, China], and miR-340 inhibitor and its control was stained with Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis kit [Multi Sciences (Lianke) Biotech. Co., Ltd.], following the manufacturer's protocol, using a BD Influx Flow Cytometer & Cell Sorter and the results were analyzed using BD FACS software version 1.0 (BD Biosciences, Franklin Lake, NJ, USA).

For analysis of cell migration and invasion, Transwell chambers (8-µm pore size; EMD Millipore, Billerica, MA, USA) and Matrigel (diluted 1:9) (Corning Incorporated, Corning, NY, USA) was used. Cells were incubated at 37°C on 6-well plates for 8 h A total of 12 h subsequent to transfection, cells were starved for 12 h. For migration assay, 2×10⁵ cells were suspended in 200 µl serum-free RPMI-1640 and seeded in the top chamber, and 350 µl RPMI-1640 containing 10% FBS was added to the lower chamber. For invasion assay, following the above process the Matrigel on the top of chamber was allowed to solidify (~5 h at 37°C). Following migration or invasion assay was 24 h of incubation at 37°C in a 5% CO₂ atmosphere, after which the cells on the upper surface of the membrane were removed and fixed in 4% paraformaldehyde ~30 min, followed by staining with 0.5% crystal violet ~60 min, all at 25°C. Cells were counted using an upright microscope (Ci-L; Nikon Corporation, Tokyo, Japan; magnification, ×200).

Prior to extracting the protein, SMMC-7721 cells were incubated at 37°C on 6-well plates for 48 h following transfection. Cells were washed twice in cold PBS and lysed in radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) with protease inhibitor cocktail (Beyotime Institute of Biotechnology). The protein concentration of cell lysates was quantified using a bicinchoninic acid kit (Beyotime Institute of Biotechnology), and equal quantities (30 µg) of protein were separated using SDS-PAGE on 10% gels, followed by transfer to a polyvinylidene fluoride membrane (EMD Millipore). The membranes were blocked in 8% skimmed milk diluted with TBS with Tween-20 [Tris-HCl 24.23 g/l, NaCl 80.06 g/l (pH 7.5), and 0.1% Tween (1 ml)] at room temperature for 1 h and incubated overnight at 4°C with primary anti-SKP2 antibody (RLT-4311) or primary anti-GAPDH antibody (RLM-3215; both 1:500; Ruiyingbio, Suzhou, China). The membranes were subsequently incubated with goat anti-rabbit IgG secondary antibody (RS0002) conjugated to horseradish peroxidase (HRP; 1:5,000; Ruiyingbio) for 3 h at 25°C. The proteins were visualized using Chemiluminescent HRP Substrate (EMD Millipore). The density was measured using Image Lab™ Software version 4.1 (Bio-Rad Laboratories, Inc.), and values were normalized to the densitometric values of GAPDH in each sample.

Using online prediction software for miRNA target genes, including PicTar (http://pictar.mdc-berlin.de/), miRanda (http://www.microrna.org/microrna/home.do) and Targetscan (http://www.targetscan.org/vert_71/).

Each experiment was repeated at least three times. Data are presented as the mean ± standard deviation. Statistical analyses were performed using Student's t-test, differences among groups were analyzed using one-way analysis of variance followed by least significant difference post hoc analysis, and the associations between miR-340 expression and clinical pathological factors were analyzed using the χ² test. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS 19.0 software (IBM Corp., Armonk, NY, USA).

In order to examine the expression of miR-340 in HCC, the expression of miR-340 in the normal hepatocyte line HL-7702 and hepatocellular carcinoma cell lines Hep3B, SMMC-7721 and Bel-7402 was determined (Fig. 1). Compared with HL-7702, RT-qPCR analysis revealed that the expression of miR-340 was decreased in the three hepatocellular carcinoma cell lines (Fig. 1C). In tissues, it was observed that the miR-340 level in tumor tissues was consistently decreased compared with adjacent non-tumor tissues (Fig. 1A). It was observed that the expression of miR-340 in tumor tissues was significantly decreased compared with adjacent non-tumor tissues (Fig. 1B). Further analysis demonstrated that patients with HCC who were hepatitis B virus (HBV)⁺ or exhibited copy numbers of HBV DNA >1.0×10³ had decreased expression of miR-340. In addition, the miR-340 level was decreased in patients who with larger tumor sizes (≥5 cm) and higher TNM grades (Table I). The results of the present study demonstrated that miR-340 may be a tumor suppressor which is associated with HCC pathogenesis, and miR-340 may correlate with disease severity.

Using RT-qPCR analysis, the expression of miR-340 in SMMC-7721 following transfection was confirmed. The results of the present study demonstrated that the expression of miR-340 in SMMC-7721 cells transfected with the miR-340 mimic was increased compared with the miR-340 mimic control. Following transfection of the miR-340 inhibitor and miR-340 inhibitor control, the expression of the latter was increased compared with the former (Fig. 2A). The CCK-8 assay demonstrated that the proliferation of SMMC-7721 cells transfected with miR-340 mimic was significantly decreased compared with the miR-340 mimic control group (Fig. 2B). Simultaneously, the proliferation ratio of SMMC-7721 cells transfected with miR-340 inhibitor was significantly increased compared with the miR-340 inhibitor control group (Fig. 2C). Using flow cytometry, it was observed that the apoptosis ratio of SMMC-7721 cells transfected with miR-340 mimic was significantly increased than miR-340 mimic control group (Fig. 2D-F); however, the apoptosis ratio of SMMC-7721 cells transfected with miR-340 inhibitor was significantly decreased compared with the miR-340 inhibitor control (Fig. 2G-I). The results of the present study demonstrated that miR-340 was able to repress cell proliferation and induce apoptosis.

Using a Transwell invasion assay, it was observed that the migration of SMMC-7721 cells with miR-340 mimics significantly decreased compared with the miR-340 mimic control group, while the migration of SMMC-7721 cells transfected with miR-340 inhibitor increased compared with the miR-340 inhibitor control group (Fig. 3A-C). Following injection of the Matrigel into the chamber, the invasiveness of SMMC-7721 cells transfected with miR-340 mimic was decreased compared with the miR-340 mimic control group. In addition, the invasiveness of cells transfected with miR-340 inhibitor was increased compared with the miR-340 inhibitor control group (Fig. 3D-F). Therefore, miR-340 was able to inhibit migration and invasion in the SMMC-7721 cell line.

Online prediction software indicated that SKP2, which is associated with cell proliferation and apoptosis, is a target gene of miR-340. Bioinformatic analysis demonstrated that the SKP2 3′ untranslated region (UTR) contains a seed sequence which complements miR-340. This suggested that miR-340 may bind strongly to the SKP2 3′UTR (Fig. 4A). Using RT-qPCR analysis, it was observed that the level of SKP2 mRNA was decreased following transfection of miR-340 mimic into SMMC-7721 cells; by contrast, transfecting miR-340 inhibitor into SMMC-7721 cells was able to increase the level of SKP2 mRNA (Fig. 4B). The protein level of SKP2 was detected by western blotting (Fig. 4C). Compared with the corresponding control group, miR-340 mimic inhibited the level of SKP2; by contrast, miR-340 inhibitor increased the level of SKP2 (Fig. 4D). Therefore, miR-340 was able to influence the expression of SKP2.