The human hepatocellular carcinoma cell lines PLC/PRF/5 and MHCC97L, and the human immortalized liver cell line HL-7702 were purchased from the American Type Culture Collection. All cells were grown in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc.) supplemented with 10% (v/v) fetal bovine serum (FBS), and incubated in a 95% humidified incubator (5% CO₂, 37°C).

PLC/PRF/5 cells (5×10⁴ cells per well) were seeded into 6-well plates and incubated in standard conditions (5% CO₂, 37°C), 24 h prior to cell transfection. Subsequently, miR-125a-5p mimic (5′-UCCCUGAGACCCUUUAACCUGUGA-3′), mimics control (5′-UUCUCCGAACGUGUCACGUTT-3′), TRIAP1-plasmid, BCL2L2-plasmid, miR-125a-5p mimic + TRIAP1-plasmid or miR-125a-5p mimic + BCL2L2-plasmid (Biovector NTCC Inc.) were transfected into the PLC/PRF/5 cells using 30 µl Lipofectamine 2000^™ reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. A total of 0.67 µg (50 pmol) miR-125a-5p mimic or mimic control, 25 µl TRIAP1-plasmid solution (0.12 g/l) or BCL2L2-plasmid solution (0.12 g/l) was used for transfection. Following incubation for 48 h, cells were ready for subsequent experimentation. Cells without any treatment were considered as the control group (Control).

Total RNA was extracted from the cells using TRIzol^® reagent (Invitrogen^™; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The RNAs were reverse transcribed to synthesize the cDNAs using PrimeScript RTreagent kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. Subsequently, qPCR was performed to analyze the cDNAs using a TaqMan Universal PCR Master Mix kit (Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 95°C for 10 min, followed by 38 cycles of 95°C for 10 sec and 58°C for 60 sec. The primer sequences were as follows: U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; GAPDH forward, 5′-GAAGGTGAAGGTCGGAGTC-3′ and reverse, 5′-GAAGATGGTGATGGGATTTC-3′; miR-125a-5p forward, 5′-CGATTCCCTGAGACCCTTTAA-3′ and reverse, 5′-TATGGTTTTGACGACTGTGTGAT-3′; TRIAP1 forward, 5′-TATCTTGCAGGAACTGTGTGCTA-3′ and reverse, 5′-AATTTAGGTTCTTCCTCCACAGC-3′; BCL2L2 forward, 5′-TGAGTTCGAGACCCGCTTC-3′ and reverse, 5′-AAAAGTTCATCGGAGACCTG-3′. GAPDH and U6 were used as the internal controls for mRNA and miRNA expression, respectively. The 2^−ΔΔCq method (16) was used to quantify relative gene expressions.

Cells were harvested and lysed using radio immunoprecipitation assay buffer containing protease inhibitor (Abcam). The concentration of protein samples was determined with a bicinchoninic acid Protein Assay kit (Bio-Rad Laboratories, Inc.). Equal amount of proteins (30 µg per lane) were separated via SDS-PAGE on 12% gels then transferred on polyvinylidene fluoride membranes (Bio-Rad Laboratories, Inc.). After blocking with 5% non-fat milk at room temperature for 2 h, the membranes were blotted overnight with the primary antibodies anti-TRIAP1 (cat no. KL507313; Kanglang Biotechnology Co., Ltd.; 1:2,000) and anti-BCL2L2 (cat no. ab38629; Abcam; 1:500) at 4°C. The next day, membranes were incubated with anti-rabbit horseradish peroxidase-linked IgG secondary antibody (cat no. 7074; Cell Signaling Technology, Inc.; 1:2,000) at room temperature for 4 h. Finally, protein bands were visualized using the enhanced chemiluminescence detection system (Super^™ Signal West Dura Extended Duration substrate; Thermo Fisher Scientific, Inc.) with the intensity analyzed with Image J software (version 1.8.0; National Institutes of Health) with GAPDH as the loading control.

MTT assay was used to assess cell viability. Briefly, PLC/PRF/5 cells were harvested and then reseeded into 96-well culture plates and cultured at 37°C for 24, 48 or 72 h. Subsequently, MTT solution (in thiazolyl blue tetrazolium bromide; Amresco LLC) was added into each culture well and incubated for a further 4 h. Finally, the optical density values were measured at 570 nm using a Synergy^™−2 Multi-function microplate reader (Bio Tek Instruments, Inc.).

Following 48 h of incubation, 3×10³ cells/well were seeded into 96-well plates and labeled with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI; Cell Signaling Technology, Inc.), as per as the manufacturer's protocol. The BD LSRFortessa X-20 flow cytometer (BD Biosciences) with FlowJo software (version 10.0.6; BD Biosciences) was used to analyze cell apoptosis.

The transfected PLC/PRF/5 cells (1×10⁵) in serum-free medium were placed into the upper chamber of a 24-well plate with 8 mm diameter (Corning, Incorporated). A total of 500 µl DMEM containing 10% FBS was added in the bottom chamber, and the cells were subsequently incubated at 37°C for 48 h. The cells in the upper chamber were removed using a cotton swab, and the membranes were stained with 0.1% crystal violet for 20 min at room temperature. Finally, the cells were counted in 10 random fields under a light microscope (magnification, ×200).

TargetScan bioinformatics software (www.targetscan.org) was used to predict the potential targets of miR-125a-5p, the findings revealed that TRIAP1 and BCL2L2 were potential targets of miR-125a-5p. To confirm this prediction, PLC/PRF/5 cells (5×10⁴ cells) were seeded into each well of a 24-well plate. After 24 h, the cells were co-transfected with TRIAP1/BCL2L2 3′-UTRpmirGLO plasmid (BioVector NTCC Inc), containing mutant TRIAP1/BCL2L2 3′-UTR or wild type TRIAP1/BCL2L2 3′-UTR, and miR-125a-5p mimic or mimic control using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Following incubation at 37°C for another 48 h, the luciferase activity (Luciferase Reporter Assay Kit; AmyJet Scientific, Inc.) was assessed using the dual-luciferase reporter assay system (Promega Corporation) and Renilla luciferase was used as the internal control for the normalization of results.

All experiments were repeated at least three times. Data are presented as the mean ± standard deviation. Statistical significance was assessed using SPSS 19.0 statistical software (IBM Corp.). Differences between multiple groups were assessed by one way analysis of variance followed by Tukey's post hoc test and differences between two groups were analyzed with Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

The expression of miR-125a-5p was detected in the human hepatocellular carcinoma cell line, PLC/PRF/5, and the human immortalized liver cell line, HL-7702, using RT-qPCR. The results indicated that, compared with the HL-7702 cells, the level of miR-125a-5p was markedly downregulated in PLC/PRF/5 cells and MHCC97L cells. The decrease of miR-125a-5p expression in PLC/PRF/5 cells was greater than in MHCC97L cells (Fig. 1), therefore, PLC/PRF/5 cells were selected for subsequent experimentation.

TargetScan database was used to predict the potential targets of miR-125a-5p. TRIAP1 and BCL2L2 were revealed to be the predicted targets of miR-125a-5p (Fig. 2A and B). To investigate whether TRIAP1 and BCL2L2 are direct targets of miR-125a-5p, luciferase reporter assay was performed. The results indicated that miR-125a-5p markedly reduced the luciferase activity of TRIAP1-3′-UTR and BCL2L2-3′-UTR in PLC/PRF/5 cells, but exerted no effect on the mutant form of TRIAP1-3′-UTR and BCL2L2-3′-UTR (Fig. 2C and D), indicating that TRIAP1 and BCL2L2 are direct targets of miR-125a-5p in PLC/PRF/5 cells.

The human hepatocellular carcinoma cell line, PLC/PRF/5 and the human immortalized liver cell line, HL-7702 were analyzed to determine the protein and mRNA expression levels of TRIAP1 (Fig. 3A) and BCL2L2 (Fig. 3B) using western blot assay and RT-qPCR respectively. These experiments demonstrated that the protein expression levels of TRIAP1 and BCL2L2 were markedly higher in the PLC/PRF/5 cells compared with the HL-7702 cells (Fig. 3A and B).

RT-qPCR analysis determined the upregulation of miR-125a-5p in PLC/PRF/5 cells, miR-125a-5p was overexpressed using miR-125a-5p mimic. After 48 h of cell transfection, the effective upregulation of the mRNA levels of miR-125a-5p as compared with the non-transfected and control-transfected cells was confirmed as observed in Fig. 4A. As shown in Fig. 4B and C, compared with the control group, the TRIAP1 was successfully overexpressed in TRIAP1 plasmids and BCL2L2 expression was up-regulated in BCL2L2 plasmids. To determine the effect of miR-125a-5p on hepatocellular carcinoma cell viability, MTT assay was performed. The results obtained revealed that the miR-125a-5p notably reduced the cell viability, but the inhibition was cancelled out upon TRIAP1 or BCL2L2 overexpression (Fig. 4D). Similarly, Transwell assay revealed that transfection with miR-125a-5p mimic markedly reduced the migration capacity of the PLC/PRF/5 cells compared with transfection with the control, but this effect was reversed when TRIAP1 or BCL2L2 were overexpressed (Fig. 4E).

Annexin V-FITC/PI apoptosis detection kit was used to determine the extent of apoptosis of cells in the different groups. Compared with the control group, the rate of cell apoptosis was markedly enhanced in the miR-125a-5p mimic transfected PLC/PRF/5 cells (Fig. 5A). Furthermore, the effect of miR-125a-5p mimic on PLC/PRF/5 cell apoptosis was demonstrated to be reversed by TRIAP1 or BCL2L2 overexpression (Fig. 5B).

A previous study indicated that TRIAP1 inhibition could induce cell apoptosis by upregulating caspase-9 and apoptotic protease-activating factor 1 (APAF1) (17). Therefore, whether caspase-9 and APAF1 were involved in regulating PLC/PRF/5 cell apoptosis by miR-125a-5p was investigated using western blots. The results demonstrated that the protein levels of caspase-9 and APAF1 were markedly increased in miR-125a-5p transfected PLC/PRF/5 cells, and this effect was inhibited by either TRIAP1 or BCL2L2 overexpression (Fig. 6A and B).