A total of nine CRC tumor tissue samples and three normal tissue samples were collected from The Third Xiangya Hospital of Central South University (Hunan, China). The present study was approved by the Independent Ethical Committee of Central South University. The samples were stored at −80°C until used. A total of five CRC cell lines, HT-29, SW480, SW620, Caco-2 and HCT-116, (American Type Culture Collection, Rockville, MA, USA) were used. The cells were grown routinely in H-Dulbecco's modified Eagle's medium (DMEM; Gibco Life Technologies, Carlsbad, CA, USA), containing 10% fetal bovine serum (Gibco Life Technologies) and cultured at 37°C in humidified air of 5% CO₂.

Rabbit polyclonal anti-EGFR antibody (1:200) was purchased from Abcam (Hong Kong, China) and Cetuximab (100 μg/ml⁻¹; 2 mg/ml⁻¹) was purchased from Merck Pharma GmbH (Erbitux, Darmstadt, Germany).

The total RNA was extracted from cells and the patient samples using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and ~500 ng extracted total RNA was reverse-transcribed into cDNA using the Primer Script RT reagent kit (Takara Bio, Inc., Otsu, Japan). The relative mRNA expression levels of EGFR were detected using a SYBR-Green qPCR assay (Takara Bio, Inc.) performed on an ABI Prism 7700 (Applied Biosystems, Foster City, CA, USA). β-actin was used as a control for normalization. The specific primer sequences used were: EGFR, forward 5′-CTTCACACATACTCCTCCTC-3′ and reverse 5′-TCTCCATCACTTATCTCCTT-3′; β-actin, forward 5′-AGGGGCCGGACTCGTCATACT-3′ and reverse 5′-GGCGGCACCACCATGTACCCT-3′. The relative expression levels of miR-133b were determined using an mirVana™ qRT-PCR miRNA Detection kit (Ambion, Austin, TX, USA), according to the manufacturer's instructions. The specific primers for miRNA-133b and U6 were purchased from GeneCopoeia, Inc. (Rockville, MD, USA). The expression levels of U6 were used as an endogenous control. All experiments were performed in triplicate and the relative expression levels were calculated using the 2^−ΔΔCt method.

The 3′UTR of the wild-type EGFR (position 50–56 of EGFR 3′UTR; GGACCAA), and a variant containing mutations (GCAGCTA) in the putative binding site, were inserted downstream of the firefly luciferase reporter into the psiCHECK-2 vector (Promega, Madison, WI, USA). The corresponding mutant construct was created by mutating the seed regions of the miR-133b binding sites and was termed 3′-UTR mut EGFR. The primer sequences used were: 3′UTR EGFR, forward 5′-CCGCTCGAGCCACGGAGGATAGTAT GAG-3′ and reverse 5′-GTTGCGGCCGCGGAAGCCTT GAAGCAGAAC-3′; 3′-UTR mut EGFR, forward 5′-GTTGCG GCCGCCCACGGAGGATAGTATGAG-3′ and reverse 5′-CCGCTCGAGGGAAGCCTTGAAGCAGAAC-3′. The cells were transfected using lipofectamine 2000 (Invitrogen Life Technologies) and were co-transfected with reporter constructs with pre-miR-133b, anti-miR-133b, pre-scramble or anti-scramble. An untreated group was used as a control. Luciferase activity was detected 48 h after transfection using a dual-luciferase reporter gene assay kit (Promega), and the luciferase activities in each group were determined using an LD400 luminometer (Promega.). Renilla luciferase activity was normalized to firefly luciferase activity. Flow cytometery was performed to determine cell apoptosis using an Annexin V-fluoresecin isothiocyanate (FITC) Apoptosis Detection kit (BD Biosciences, Frankin Lakes, New Jersey, United State). At 24 h post-transfection, the cells were harvested and washed twice with cold PBS. Subsequently, 10⁶ cells were resuspended in 200 μl binding buffer with 10 μl Annexin-FITC and 5 μl propidium iodide, followed by incubation in the dark for 30 min. Finally, 300 μl binding buffer was added and the cells were assessed using flow cytometric analysis (Moflo XDP; Beckman Coulter, Brea, CA, USA).

The cells were seeded into 96-well plates at a density of 5×10³ cells/well and were incubated for 24, 48 and 72 h at 37°C with 5% CO₂. The viability of the cells was assessed using an MTT assay. Briefly, MTT (10 mg/ml) was added to the cells and incubated for 3 h at 37°C The reaction was terminated by removal of the supernatant, followed by the addition of 200 μl dimethyl sulfoxide and gently pipetting. Following a 2 h incubation, the optical density of each well at 570 nm was measured with a BioRad microplate reader (Bio-Rad, Hercules, CA, USA). Each assay was performed in triplicate.

The cells were digested, washed with phosphate-buffered saline (PBS), and subsequently fixed in 70% ethanol at 4°C overnight. The fixed cells (1×10⁶) were washed with PBS and resuspended in staining solution, containing 50 μg/ml propidium iodide, 1 mg/ml RNase A and 0.1% Triton X-100 in PBS. Following incubation for 30 min at 4°C, the stained cells were analyzed on a flow cytometer (Beckman Coulter).

Cell invasion was determined using a transwell assay. Briefly, the cells were suspended in serum-free medium and aliquots (1×10⁵ cells) of the prepared cell suspension were added into the upper chamber. The lower chamber was filled with 1 ml DMEM, containing fetal bovine serum (10%). Following incubation for 24 h at 37°C, the cells remaining on the upper side of the membrane were removed using a cotton swab, while the cells, which had migrated through the membrane were fixed with 75% alcohol and stained with 1% crystal violet for 20 min. The invasive cells were subsequently counted and images were captured using an inverted microscope (Nikon, Tokyo, Japan).

The present study used TALEN technology to knock out the EGFR gene in Caco-2 cells. TALENs designed to target the EGFR gene were purchased from Sidansai Biotechnology (Shanghai, China). The cells in 24-well plates were transfected with 400 ng TALEN expression plasmids (EGFR-TALEN) or negative control (TALEN-NC) using Lipofectamine 2000 (Invitrogen Life Technologies), according to the manufacturer's instructions. Western blotting was performed to determine the protein expression levels of EGFR and confirm the efficiency of the TALEN mediated knockout.

To determine the potential clinical and pathological implications of an altered expression of miR-133b in CRC tumor samples, the present study investigated the expression levels of miR-133b in nine CRC tumor samples and three normal tissue samples using RT-qPCR. As shown in Fig. 1A, the expression levels of miR-133b were significantly lower in CRC tumor samples compared with the normal tissue samples. Furthermore, the abundance of miR-133b in five CRC cell lines was demonstrated. Compared with normal colonic mucosa epithelial cells isolated and pooled from three adjacent non-cancerous tissue samples, the expression levels of miR-133b in the five CRC cell lines were significantly reduced to different extents, markedly lower in the SW480 and Caco-2 cells (Fig. 1B). These results suggested that the expression levels of miR-133b were significantly decreased in human CRC specimens and cell lines, which suggested that it may be involved in the pathogenesis of CRC.

To determine whether miR-133b directly targets EGFR in CRC cells, a luciferase assay was performed. A region of 501 bp length of the wild-type 3′-UTR of EGFR, containing the predicted miR-133b target sites, was amplified and inserted into the resulting amplicon downstream of a lucife rase reporter gene (termed wt-EGFR). A mutant version of the 3′-UTR of EGFR lacking the miR-133b binding sites (named mut-EGFR), was also constructed. The SW480 and Caco-2 cells were co-transfected with wt-EGFR or mut-EGFR vectors and either the miR-133b mimics (pre-miR-133b), miR-133b inhibitor (anti-miR-133b) or their respective scrambled controls (pre-scramble and anti-scramble). The results showed that miR-133b significantly suppressed the luciferase activity of the reporter gene, containing the 3′UTR of EGFR compared with the controls, however, this was significantly rescued when the miR-133b binding sites were absent in the SW480 and Caco-2 cells (Fig. 2A and B). Furthermore, the inhibitory effect of miR-133b on EGFR expression was assessed. RT-qPCR analysis demonstrated that an sincreased expression of miR-133b significantly decreased the mRNA expression levels of EGFR compared with the cells transfected with the control (Fig. 2C and D). Taken together, these results confirmed that miR-133b directly targeted EGFR and repressed its expression levels in CRC cells.

To investigate the roles of miR-133b in the CRC cells, the expression of miR-133b was restored in SW480 and Caco-2 cells. This resulted in a lower expression of miR-133b in the selected CRC cell lines, by liposomal delivery. The effect of miR-133b on cell growth and invasion was investigated. An MTT assay demonstrated that the overexpression of miR-133b decreased the cell viability in SW480 and Caco-2 cells (Fig. 3A and B). It was also revealed that miR-133b marginally increased the percentage of G1 phase cells in SW480 and Caco-2 cells (Fig. 3C and D), as determined by flow cytometric analysis. A Transwell assay indicated the forced expression of miR-133b significantly inhibited the cell invasion ability compared with the control group in SW480 and Caco-2 cells (Fig. 3E and F). These results suggested that miR-133b, which serves as a tumor suppressor, is important in the control of growth and invasion of CRC cells in vitro.

Based on the above finding that miR-133b inhibited the cell growth and invasion, at least in part, by downregulating the expression levels of EGFR, the present study hypothesized that inhibiting the expression of EGFR by other approaches would also have similar inhibitory effects. EGFR was knocked out using TALEN technology, a highly effective approach for the targeted knockout of genes in mammalian cells. Western blotting was performed to confirm the TALEN-mediated knock-out efficiency in Caco-2 cells. As shown in Fig. 4A, the EGFR gene was silenced effectively in the EGFR-TALEN vector transfected cells. As expected, silencing of EGFR by TALEN decreased the cell viability (Fig. 4B), increased the percentage of cells in the G1 phase (Fig. 4C) and inhibited the invasiveness (Fig. 4D) of Caco-2 cells. This suggested that EGFR silencing may be a common effective strategy to inhibit the growth and invasion in CRC cells.

Cetuximab is a chimeric monoclonal antibody, which binds to EGFR and competitively inhibits ligand binding to suppress tumor proliferation. Cetuximab has been approved and is used for the treatment of patients with CRC. The present study aimed to determine whether the combination of miR-133b and cetuximab provided an improved antitumor effect in CRC cells. To carry this out, the SW480 and Caco-2 cells were treated with cetuximab following transfection with the miR-133b mimics or scramble control. Notably, the combination treatment of miR-133b and cetuximab exhibited an increased inhibitory effect on the cell viability (Fig. 5A and B) and invasion (Fig. 5E and F) of the SW480 and Caco-2 cells compared with treatment with either miR-133b or cetuximab alone. It was also demonstrated that combinational treatment had a marginally increased inhibitory effect on cell cycle progression into S phase, as determined by flow cytometric analysis (Fig. 5C and D). These data suggested that this was a promising option of a combinational therapy for patients with CRC.