The human HepG2 HCC cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from GE Healthcare Life Sciences (Beijing, China). 5-FU and DDP were purchased from Sigma-Aldrich (St. Louis, MO, USA). The miR mimics were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China).

The miRanda (http://www.microrna.org/), TargetScan (http://www.targetscan.org/vert_61/), Pictar (http://pictar.mdc-berlin.de/cgi-bin/PicTar_vertebrate.cgi) and miRBase (http://www.mirbase.org/search.shtml) algorithms were used to predict the putative target genes of miR-133a and miR-326.

The human HepG2 HCC cell line was maintained in DMEM, supplemented with 4.5 g/l glucose, 10% FBS and 1% penicillin/streptomycin (GE Healthcare Life Sciences) in a humidified atmosphere containing 5% CO₂ at 37°C.

The full-length Bcl-xl cDNA construct containing the entire 3′UTR was synthesized and cloned into a pcDNA3.1 plasmid (Landbiology, Guangzhou, China). Site directed mutagenesis using the QuikChange Site-Directed Mutagenesis kit (Agilent Technologies GmbH, Waldbronn, Germany) using the following primers (Shanghai Novland Co., Ltd., Shanghai, China): Forward, 5′-GCTCCCATGACCATACTGAGCCTGGTTCTGGGCCCAAGACAGATGCC-3′ and reverse 5′-GGCATCTGTTCTTGGGCCCAGAACCAGGCTCAGTATGGTCATGGGAGC-3′. The 3′UTR (wild-type and mutant) of Bcl-xl was synthesized and was inserted downstream of the Renilla luciferase gene of the dual-luciferase. The primer for Bclxl was designed, and targeted fragments were obtained by PCR amplification (Stratagene, Santa Clara, CA, USA). Subsequently, the specificity of the PCR primers was analyzed through digesting PCR products with restriction endonuclease. The target gene was then linked with the vector and recombinant plasmids were amplified by transformation into E. coli (Landbiology). Positive clones were selected randomly and sequenced for verification.

The wild-type and mutant Bcl-xl 3′UTR-luciferase reporter plasmids were co-transfected with a miR-133a or miR-326 mimic (Shanghai GenePharma, Co., Ltd.) into the HEK923 cells (American Type Culture Collection, Manassas, VA, USA) using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA). At 48 h post-transfection, the cells were assayed for luciferase activity using a Dual-Luciferase Assay system (Promega Corporation, Madison, WI, USA), according to the manufacturer's instructions. The Renilla luciferase activities were normalized to the corresponding activities of firefly luciferase. For each transfection, the average luciferase activity was calculated from three replicates.

RT-qPCR was performed, as previously reported (24). Total RNA was extracted from the cells using TRIzol reagent (Tiangen Biotech Co., Ltd., Beijing, China), according to the manufacturer's instructions. The primer sequences for Bcl-xl, Bax, GAPDH, miR-133a, miR-326 and U6 were as follows: Bcl-xl, forward 5′-GCTGGTGGTTGACTTTCTCTCCTAC-3′ and reverse 5′-CCTCAGTCCTGTTCTCTTCCACATC-3′; Bax, forward 5′-CCCTTTTCTACTTTGCCAGCA-3′ and reverse 5′-GGAGTCTCACCCAACCACCC-3′; GAPDH, forward 5′-CATGAGAAGTATGACAACAGCCT-3′ and reverse 5′-AGTCCTTCCACGATACCAAAGT-3′; miR-133a, forward 5′-TCATATTTGGTCCCCTTCAACC-3′ and reverse 5′-TATCGTTGTTCTCCACTCCTTCAC-3′; miR-326, forward 5′-ACTGTCCTTCCCTCTGGGC-3′ and reverse 5′-AATGGTTGTTCTCCACTCTCTCTC-3′; and U6 small nuclear RNA, forward 5′-ATTGGAACGATACAGAGAAGATT-3′ and reverse 5′-GGAACGCTTCACGAATTTG-3′. The U6 small nuclear RNA was used as an internal control for miR quantification. To determine the mRNA expression levels of Bcl-xl and Bax, the total RNA were reverse transcribed into cDNA using a Custom Reverse Transcription kit. The qPCR analyses of Bcl-xl and Bax were performed using the above-mentioned primers, and normalized to GADPH. SYBR green qPCR was performed on a MX3000P PCR System (Agilent Technologies, Inc., Santa Clara, CA, USA). The cycling programme consisted of 40 cycles, with each cycle consisting of 3 min at 95°C, 12 sec at 95°C and 40 sec at 62°C. The relative expression ratios of Bcl-xl, Bax, miR-133a, miR-326 were calculated using the 2^−ΔΔCt method (25).

The cells were transfected using Lipofectamine 2000 transfection reagent according to the manufacturer's instructions (Invitrogen Life Technologies) The sequences for the two miRNA mimics were as follows: miR-133a: 5′-uuugguccccuucaaccagcug-3′; miR-326: 5′-ccucugggcccuuccuccag-3′. At 1 day prior to transfection, the cells were plated in the appropriate quantity of growth medium without antibiotics in order to reach 80–90% confluency at the time of transfection. For each transfection sample, miRNA mimics- Lipofectamine 2000 complexes were prepared as follows: miRNA mimics (Shanghai GenePharma, Co., Ltd.) were diluted in the appropriate quantity of Opti-minimum essential medium (MEM; Gibco-BRL, Grand Island, NY, USA) without serum and mixed gently. Lipofectamine 2000 was diluted in the appropriate quantity of Opti-MEM without serum then mixed gently. The samples were incubated for 5 min at room temperature. The diluted miRNA mimics were then combined with the diluted Lipofectamine 2000 and incubated for 20 min at room temperature to allow complex formation to occur. The miRNA mimics-Lipofectamine 2000 complexes were added to each well containing cells and medium. The cells were incubated at 37°C in a CO₂ incubator until harvesting of cells and the assay for the target gene.

At 48 h post-transfection, the cell lysates were prepared by lysis in radioimmunoprecipitation assay buffer (CWBIO, Beijing, China) with protease inhibitors. Centrifugation was then performed at 10,000–14,000 × g for 15 min at 4°C, and the protein concentration was determined using a bicinchoninic acid assay (CWBIO). The proteins (30–50 µg) were separated on 10% SDS-polyacrylamide gels (CWBIO) and were transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). A PageRuler prestained protein ladder (CWBIO) was used as a molecular marker. A single membrane was cut into three parts at the 43 kD, 30 kD and 20 kD bands, and incubated with anti-β-actin (1:1,000; cat. no. 12620), anti-Bcl-xl (1:1,000; cat. no. 2764) and anti-Bax (1:1,000; cat. no. 5023) monoclonal rabbit primary antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA), respectively. The proteins were detected using a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:4,000; cat. no. CW0103; CWBIO) and a ChemiLucent ECL Detection system (EMD Millipore, Billerica, MA, USA). Quantification of the protein bands was performed using AlphaImager 2200 software (National Institutes of Health, Bethesda, MD, USA).

The HepG2 cells in the exponential growth phase were seeded into 96-well plates at a density of 4×10³ cells/well and were incubated for 12 h at 37°C. The cells were transfected with either the miR-133a mimic, miR-326 mimic or the negative control (NC). At 24 h post-transfection, various concentrations of 5-FU (0, 5, 50, 500 and 5,000 µM), or DDP (0, 6.25, 12.5, 25, 50 and 100 µM) were added. Subsequent to incubation for 24 h, 20 µl MTT (Amresco LLC, Solon, OH, USA) solution, at a concentration of 5 mg/ml, was added to each well for 4 h at 37°C. The culture medium was then removed, and the insoluble formazan crystals were dissolved in 150 µl dimethyl sulf-oxide (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China). Following agitation for 10 min, the absorbance at 490 nm (A490) was optically monitored using a 318-microplate reader (Shanghai Sanco Instrument Co., Ltd., Shanghai, China).

SPSS software, version 18.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. All values are expressed as the mean ± standard deviation. One-way analysis of variance was performed to evaluate the significance of the differences between samples. P<0.05 was considered to indicate a statistically significant difference.

To examine the mechanism of miR-133a and miR-326, the TargetScan, Pictar, miRanda and miRBase computational programs were used to predict putative terget genes for the two miRs. The programs predicted the common target gene for miR-133a and miR-326, as the human Bcl-xl gene (Fig. 1).

The present study confirmed the predicted binding sites recognized by miR-133a and miR-326 using the pmirGLO dual-luciferase vector. The wild-type, containing binding sited with the Bcl-xl3′UTR or mutant, containing binding sites with deletion of the Bcl-xl were cloned downstream of firefly luciferase using the pmirGLO vector and were co-transfected with miR-133a or miR-326 in the HEK293 cells. As demonstrated in Fig. 2, in the cells transfected with the vector containing the Bcl-xl 3′UTR fragment with the binding site for Bcl-xl, the luciferase activity was significantly inhibited following transfection of miR-133a compared with the cells that were transfected with the NC miR mimics and untreated cells. Similar results were observed in the cells transfected with miR-326.

Using RT-qPCR, the differences in the expression levels of miR-133a and miR-326 between the mimic-transfected cells and untreated cells were assessed. The miR expression levels in the HepG2 cells transfected with the miR-133a or miR-326 mimics were significantly upregulated compared with the control cells and the untreated cells (Fig. 3).

RT-qPCR was performed to assess whether miR-133a and miR-326 affected the mRNA expression of Bcl-xl. The data demonstrated that transfection with either miR-133a or miR-326 reduced the mRNA expression levels of Bcl-xl compared with the negative control group, whereas the expression levels of Bax were increased, as expected (Fig. 4). This suggested that mRNA degradation may be an important mechanism underlying the miR-133a and miR-326-mediated post-transcriptional regulation of Bcl-xl.

In order to examine the effect of miR-133a and miR-326 on the endogenous expression of Bcl-xl, the miR-133a, miR-326 or negative control mimics were transfected into HepG2 cells. The results demonstrated that enhanced expression of miR-133a or miR-326 significantly reduced the protein expression levels of Bcl-xl compared with negative control and mock-transfected cells, whereas the protein levels of Bax exhibited the opposite effects (Fig. 5), consistent with the changes in mRNA levels.

To investigate whether miR-133a and miR-326 sensitize HepG2 cells to 5-FU and DDP, the miR-133a mimic, miR-326 mimic or negative control plasmids were transfected into the HepG2 cells. An MTT cell proliferation assay was performed to determine the rate of cell survival. The results indicated that transfection with the miR-133a or miR-326 mimics significantly reduced cell viability compared with negative control cells and the mock-transfected cells (Fig. 6).