Pter (purity, >99%; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany).

The SMMC-7721 HCC cell line (China Center for Type Culture Collection, Wuhan University, Wuhan, China) was cultured and maintained in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) with 100 U/ml penicillin and 100 U/ml streptomycin. Cells were incubated at 37°C with 5% CO₂.

SMMC-7721 cells at a density of 60–70% confluence were divided into the following five groups: Pter treatment group (50 µM Pter), miR-19a inhibitor group (transfected with miR-19a inhibitor 5′-UCAGUUUUGCAUAGAUUUGCACA-3′), Pter + miR19a inhibitor group (50 µM Pter and transfected with miR-19a inhibitor), negative control group (transfected with negative control 5′-UUGUACUACACAAAAGUACUG-3′) and the blank group (DMSO treatment) at 37°C for 48 h. Lipofectamine 2000 (5 µl; Invitrogen; Thermo Fisher Scientific, Inc.) was diluted to 100 µl in serum-free RPMI-1640 medium. miR-19a inhibitor (20 pM) or negative control (20 pM) was also diluted to 50 µl in the serum-free RPMI-1640 medium at room temperature for 5 min. Both of the dilutions were subsequently mixed at room temperature for 20 min prior to transfer into culture plates for cell transfection at 37°C in 5% CO₂ for 6 h. The transfected cells were subsequently cultivated in complete medium (RPMI-1640 medium containing 10% FBS with 100 U/ml penicillin and 100 U/ml streptomycin) for 48 h and then the subsequent experiments including qPCR, western blotting, MTT and cell cycle and apoptosis assay were performed.

Following treatment of cells (5×10⁷ cells/well) with Ctrl (DMSO), 5, 25, 50 and 100 µM Pter at 37°C for 24 h, and the establishment of the 5 treatment groups described above, the total RNA was extracted from SMMC-7721 cells using TRIzol (Life Technologies; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. RNA concentration was detected at an absorbance of A₂₆₀ and A₂₈₀. cDNA was synthesized by using a PrimeScript RT reagent kit (Takara Bio, Inc., Otsu, Japan). The PCR amplification primer sequences are presented in Table I. qPCR was performed by the MiniOpticon Real-Time PCR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using SYBR Premix Ex Taq (Takara Bio, Inc.). Following initial denaturation at 95°C for 30 sec, amplifications were performed for 40 cycles at 95°C for 5 sec and 60°C for 30 sec. miR-19a levels were compared to U6 as an internal reference and PTEN was compared with β-actin. qPCR was repeated 3 times. The relative expression of miRNA and mRNA was calculated by the 2^−ΔΔCq method (26).

Following treatment of cells (5×10⁴ cells/well) with Ctrl (DMSO), 5, 25, 50 and 100 µM Pter at 37°C for 24 h, and establishment of the 5 treatment groups described above, whole proteins were extracted by RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) from SMMC-7721 cells which was quantified by a BCA Protein Assay kit (Beyotime Institute of Biotechnology) and separated at 20 µg/lane by 15% SDS-PAGE prior to transfer onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.) using an Electrophoresis Transfer System (Bio-Rad Laboratories, Inc.). The blots blocked with 5% fat-free milk in a Tris-buffered saline with 5% Tween 20 (TBST) for 1 h at room temperature, were probed with anti-PTEN (mouse monoclonal; cat. no. ab79156; 1:500; Abcam, Cambridge, UK), anti-Akt (Rat polyclonal; cat. no. ab8805; 1:500; Abcam), anti-phosphorylated (p)-Akt (Rat polyclonal; cat. no. ab8933; 1:500; Abcam) and anti-β-actin (Rat polyclonal; cat. no. ab8227; 1:2,000; Abcam) primary antibodies overnight at 4°C, and subsequently treated with the respective secondary antibodies (Rat, cat. no. ab218695; 1:4,000 or mouse cat. no. ab131368; 1:4,000 dilution; Abcam) for 1 h at 4°C. The proteins were visualized by using an enhanced chemiluminescence detection kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Densitometric analysis was performed to quantify protein expression using ImageJ software version 1.49 (National Institutes of Health, Bethesda, MD, USA).

Following treatment of cells (5×10⁴ cells/well) with Ctrl (DMSO), 20, 40, 60, 80 and 100 µM Pter at 37°C for 24, 48 or 72 h, and establishment of the five treatment groups described in the cell transfection section above, the proliferation of SMMC-7721 was detected by MTT assays (Sigma-Aldrich; Merck KGaA). To investigate cell viability, 0.5 mg/ml MTT solution was added into each well following seeding of the treated cells for 24 h at a density of 3×10⁵ cells/ml per well. Cells were subsequently incubated for 4 h at 37°C with 5% CO₂. The supernatant liquid was removed and 150 µl DMSO was added to each well. A microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) was used to determine optical density values at 570 nm after 24, 48 and 72 h. For the experiments that consisted of five different treatment groups, measurements were taken after 24 h.

Cell cycle and apoptosis was examined in the five treatment groups by flow cytometry. Annexin V-fluorescein isothiocyanate (10 µl) and propidium iodide (PI; 5 µl, Sigma-Aldrich; Merck KGaA) were incubated with the cells (5×10⁵ cells/well) in each group in the dark at 4°C for 30 min. A flow cytometer (BD Biosciences, San Jose, CA, USA) was used to calculate the percentage of apoptotic cells with FlowJo version 10 software (FlowJo LLC, Ashland, OR, USA).

To analyze the cell cycle, cells (5×10⁵ cells/well) were washed twice with PBS and fixed with 75% ethanol overnight at 4°C. The cells were subsequently stained with 5 µl PI/ribonuclease A (Sigma-Aldrich; Merck KGaA) at 4°C for 30 min in the dark. Data was analyzed with a flow cytometer (BD Biosciences). A total of 14,000 fluorescence signals for each PI-stained sample were collected and calculated by ModFit LT version 3.2 software (Verity Software House, Inc., Topsham, ME, USA).

SMMC-7721 cell invasion ability was analyzed in Transwell chambers (pore size, 8 µm; Corning Incorporated, Corning, NY, USA) coated with Matrigel in serum-free medium. Following incubation, the 5×10⁴ cells/well were added into the upper chamber at 37°C in a humidified 5% CO₂ for 24 h. Then the cells were removed from the upper chamber (medium containing serum-free RPMI-1640) and the invading cells in the lower chamber (medium containing 20% FBS) were fixed with 4% paraformaldehyde at 37°C for 15 min, then stained with 0.1% crystal violet at 37°C for 15 min and counted using a phase-contrast microscope at ×200 magnification of each membrane in 5 representative fields.

The binding sequence of PTEN 3′UTR and miR-19a was predicted by TargetScanHuman version 7.1 (www.targetscan.org/vert_71/). The pGL3-Luciferase vector with firefly and Renilla luciferase activity (Wuhan Jin Kairui Biological Engineering Co., Ltd, Wuhan, China) was digested with XbaI and the target sequence was inserted. The promoter sequence of PTEN 3′UTR containing the binding site was synthesized as the wild-type (WT) plasmid (PTEN-WT). Similarly, a mutated binding site of PTEN was constructed as the mutant-type (MT) plasmid (PTEN-MT). The cells (5×10⁴ cells/well) were transfected by using Lipofectamine 2000^® (5 µl; Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 48 h. The cells were subsequently co-transfected with the luciferase reporter plasmid (20 pM) and the miR-19a inhibitor (20 pM) or negative control (20 pM) prior to treatment with the Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol. Reporter gene activity was detected and normalized with the ratio of Renilla and firefly luciferase activity.

Data are presented as the mean ± standard deviation from three independent experiments. All statistical analyses were performed with SPSS version 21.0 software (IBM Corp., Armonk, NY, USA). And unpaired Student's t-tests were employed to analyze the difference between two groups. P<0.05 was considered to indicate a statistically significant difference.

Previous reports have indicated that the modulation of PTEN-targeting miRNAs by Pter in prostate cancer led to the overexpression of PTEN and downregulation of PI3K-Akt pathway activity (17,27). Thus, the present study aimed to confirm this hypothesis in HCC cells. qPCR was performed to measure the relative expression of miR-19a in SMMC-7721 cells. The results demonstrated that miR-19a expression was decreased by Pter treatment in a dose-dependent manner in SMMC-7721 cells, indicating that Pter may exhibit important antioncogenic activity (P<0.05; Fig. 1A). In addition, miR-19a expression was also significantly lower in the Pter, miR-19a inhibitor and Pter + miR-19a inhibitor groups, compared with the blank or negative control groups (P<0.05; Fig. 1B).

The MTT assay revealed that Pter repressed SMMC-7721 cell proliferation in a time- and dose-dependent manner (Fig. 1C). Furthermore, after 24 h, the cell viability in the miR-19a inhibitor and Pter + miR-19a inhibitor groups was markedly reduced compared with the blank or negative control group (P<0.05; Fig. 1D). Additionally, a synergistic effect was observed between Pter and the miR-19a-inhibitor. Both Pter and miR-19a inhibitor treatments significantly repressed SMMC-7721 cell proliferation (Fig. 1D). Pter exerted the most potent inhibitory effect on SMMC-7721 cell proliferation.

Flow cytometry was performed to analyze the cell cycle of the cells treated with Pter. No statistically significant differences were observed between the cells in the blank and negative control groups in each phase (P>0.05; Fig. 2A and B). However, the Pter treatment and miR-19a inhibitor groups demonstrated a significantly higher number of cells in the S phase, and significantly fewer cells in the G2/M phase, compared with the blank or negative control groups (P<0.05; Fig. 2A and B), indicating increased cell cycle arrest in S phase and reduced cell mitosis following Pter treatment or miR-19a inhibition. Furthermore, the apoptosis of cells was also analyzed; the apoptotic rate of the blank and negative control groups were not significantly different, but Pter treatment or miR-19a inhibition significantly increased the apoptotic rate compared with blank or negative control groups (P<0.05; Fig. 2C and D). Synergy between Pter treatment and miR-19a inhibitor transfection was also observed (Fig. 2C and D). Additionally, cell invasion ability was significantly decreased by Pter and miR-19a inhibitor, compared with blank or negative control groups (Fig. 3A and B).

A luciferase reporter gene assay was performed to verify whether PTEN may be a direct target gene of miR-19a. It was demonstrated that the target sequence of miR-19a and PTEN 3′-UTR matched (Fig. 3C). Therefore, the pGL3-Luciferase vector was constructed and the PTEN 3′UTR WT (AGCUUA) or MT (ACACGG) sequence was inserted downstream, and the vector was subsequently co-transfected with the miR-19a inhibitor or negative control. Following PTEN 3′-UTR-WT co-transfection with miR-19a inhibitor, luciferase activity was significantly higher compared with the negative control group; however, no statistically significant difference was observed between the negative control and miR-19a inhibitor group when co-transfected with PTEN 3′UTR-MT (Fig. 3D). There results indicated that the base sequence of miR-19a matched the PTEN mRNA 3′UTR and that PTEN is a target site of miR-19a.

Initially, western blot analysis was performed to determine PTEN protein expression following treatment with various concentrations of Pter. The results demonstrated that Pter increased PTEN protein expression in a dose-dependent manner (Fig. 4A and B). Furthermore, to investigate the association between miR-19a and PTEN expression further, SMMC-7721 cells with low expression levels of miR-19a were established by miR-19a inhibitor transfection. The Pter and miR-19a inhibitor groups exhibited significantly increased expression of PTEN mRNA compared with the blank or negative control groups, respectively (P<0.05; Fig. 4C). A synergistic effect was observed in the Pter + miR-19a inhibitor group (P<0.05; Fig. 4C).

Additionally, reduced miR-19a expression induced a marked upregulation of PTEN protein expression compared with the negative control group (Fig. 5A and B). These results indicated that Pter treatment may enhance PTEN expression via the downregulation of miR-19a.

PTEN, Akt and p-Akt levels were detected by western blot analysis. Previous research has indicated that PTEN is an inhibitor of the PI3K/Akt pathway (28). The present study revealed a negative association between PTEN and miR-19a expression, and a positive association between PTEN and Akt expression, which indicates an association between the PI3K/Akt pathway and miR-19a. In the current study, the Pter and miR-19a inhibitor groups exhibited significantly upregulated PTEN protein expression and significantly reduced Akt and p-Akt levels (P<0.05; Fig. 5A and B). These results indicate that miR-19a may mediate the downstream targets of the PTEN/Akt signal pathway to affect the biological behavior of HCC (Fig. 5C).