Cell lines MDA-MB-231 and MCF-7 were obtained from KeyGen Co. (Nanjing, China) and cultured in Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (both from Gibco, Grand Island, NY, USA) at 37°C in a humidified atmosphere under 5% CO₂.

Twenty-nine breast cancer and matched adjacent tissue samples were resected from patients at the Second Affiliated Hospital of Dalian Medical University (Dalian, China) from July 2011 to June 2014. Informed consent forms and the entire protocol were approved by the Ethics Committee of the Second Affiliated Hospital of Dalian Medical University. The samples were stored at −80°C, and total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA).

The concentration and quality of each total RNA sample was determined using A260/A280 spectrophotometric reading. cDNA was synthesized with TaqMan reverse transcription reagents (Applied Biosystems, Branchbury, NJ, USA), following the manufacturer's recommendations. Real-time PCR was carried out using 7500 Fast Real-time PCR system (Applied Biosystems). Reactions were run in 3 independent experiments. The relative expression level of FUT4 was normalized to GAPDH. The primer sequences were: 5′-TCCTACGGAGAGGCTCAG-3′ and 5′-TCCTCGTAGTCCAACACG-3′. RT-PCR for miR-493-5p was performed using Real-Time PCR Universal Reagent (GenePharma, Shanghai, China). U6 was used as an internal control.

Protein was extracted from the cells using 1X radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA), subjected to SDS-PAGE, and then transferred to polyvinylidene difluoride membranes. The membranes were blocked in 5% skimmed milk for 2 h, probed with the antibody against human FUT4 (1:1,000 dilution; Abcam, Cambridge, UK) or GAPDH (1:1,000 dilution) at 4°C overnight, and with peroxidase-conjugated secondary antibody (1:1,000 dilution) (both from Santa Cruz Biotechnology), and then visualized by chemiluminescence (GE Healthcare, Fairfield, CT, USA).

All animal experiments were performed according to the protocol of the Dalian Committee on Animal Care using 5- to 6-week-old male athymic nude mice. Cells (1×10⁷) were subcutaneously injected into the right flank of each nude mouse. The length (L) and width (W) of each tumor were measured every 7 days with calipers, and the volume was calculated.

The FUT4 coding sequence (CDS) was obtained and inserted into the NotI and BamHI sites of the pGLV5/H1/GFP+Puro lentiviral plasmid, respectively. The FUT4 shRNA sequences were inserted into the BamHI and EcoRI sites of the pGLV3/H1/GFP+Puro lentiviral plasmid. Lentiviral plasmids were co-transfected with PG-P1-VSVG, PG-P2-REV and PG-P3-RRE plasmids into 293T cells (Invitrogen), and virus-containing supernatants were prepared according to the manufacturer's instructions. For the lentiviral infection, the cells cultured in 6-well tissue culture plates were infected with the lentiviral vectors at a multiplicity of infection of 40 for 24 h. The medium was replaced with fresh complete medium. After 2 days, the cells were observed by fluorescence microscopy to confirm that >90% of the cells were GFP-positive. Subsequently, the GFP-positive cells were screened by addition of 5 µg/ml puromycin.

Cell invasion was assessed using the Matrigel invasion chamber (Corning, Corning, NY, USA) in triplicate. The cells (1.0×10⁵) were harvested in serum-free medium containing 0.1% BSA and plated to the upper chamber precoated with Matrigel. Medium containing 10% FBS in the lower chamber served as the chemoattractant. After the cells were incubated at 37°C in a humidified incubator with 5% CO₂ for 24 h, the non-invading cells were removed with cotton swabs. The invasive cells that had attached to the lower surface of the inserted membrane were fixed in 100% methanol at room temperature and stained with Wright-Giemsa. The number of invasive cells on the lower surface of the membrane was then counted under a microscope.

A pmirGLO Dual-Luciferase miRNA target expression vector was used for 3′UTR luciferase assays (Promega, Madison, WI, USA). The target genes of miRNA-493-5p were selected based on target scan algorithms (microRNA.org (http://www.microrna.org/microrna/home.do), MicroCosm (http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/) and TargetScan (http://www.targetscan.org/). For 3′UTR luciferase assay, the cells were co-transfected with hsa-miR-493-5p mimics or pmirGLO Dual-Luciferase miRNA target expression vectors and the wild-type or mutant target sequence using Lipofectamine 2000. Luciferase assay was performed using the Dual-Luciferase^® reporter assay system (Promega) after transfection at 48 h. Data are presented as the mean value ± SD for triplicate experiments.

Statistical analyses were carried out using the SPSS 17.0 program. An independent sample t-test was run to analyze the significance of the differences found. P-value <0.05 was considered to indicate a statistically significant result. All data are presented as the mean ± SD.

We first determined the expression of FUT4 in breast cancer cell lines, primary breast cancer and the matched adjacent tissue samples. As shown in Fig. 1A and B, the FUT4 (5.76-fold) expression level was significantly higher in the MDA-MB-231 cell line than that in the MCF-7 cell line. Furthermore, analysis of FUT4 expression in pairs of the primary breast cancer and matched adjacent tissue samples revealed that FUT4 was upregulated in the primary breast cancer tissue samples (Fig. 1C and D). These data support the assumption that the differential expression of FUT4 may be associated with breast cancer.

To test whether FUT4 plays a role in regulating invasion, we infected MCF-7 cells with lentiviral vectors containing FUT4. After infection, the MCF-7/FUT4 cells expressed high exogenous FUT4 (Fig. 2A and B) in comparison with the uninfected controls or GFP-expressing cells.

Overexpression of FUT4 increased the invasive ability of the MCF-7 cells in vitro (Fig. 2C). In order to further confirm that FUT4 affects invasive ability, we used shRNA delivered in a lentiviral vector tagged with GFP to regulate FUT4 expression in the MDA-MB-231 cells. Three different shRNA lentiviruses significantly reduced the FUT4 expression >60% at the mRNA level and >50% at the protein level (Fig. 3A and B). The invasive ability was significantly decreased to 55% in the MDA-MB-231/FUT4-shRNA cells compared with the invasive ability noted in the control cells (Fig. 3C).

In order to identify whether FUT4 further affects the tumorigenicity in vivo, MCF-7/control, MCF-7/FUT4, MDA-MB-231/control shRNA and MDA-MB-231/FUT4-shRNA1 were subcutaneously injected into nude mice and tumor formation was monitored. On day 28, the mice were sacrificed under anesthesia and tumor weights were measured. Tumors grew faster in the MCF-7/FUT4 and MDA-MB-231/control shRNA groups when compared with the rates in the MCF-7/control and MDA-MB-231/FUT4-shRNA1 groups, respectively (Figs. 2D and 3D). These data suggest that FUT4 enhanced the cell growth in vivo.

To understand the mechanisms by which miRNAs execute their function by FUT4, we adopted 3 bioinformatic algorithms (TargetScan, MicroCosm and miRanda) to identify a large number of potential miRNAs. Among these candidates, miR-493-5p was selected for further study. Total RNA was isolated from the cell lines, the breast cancer tissue and the matched adjacent tissue samples, and the miR-493-5p levels were determined using TaqMan real-time PCR. The miR-493-5p expression level was higher in the MCF-7 cells than that in the MDA-MB-231 cells, while the tissue samples showed the same tendency (Fig. 4A and B).

To test whether FUT4 is a direct target of miR-493-5p, luciferase activity assays were performed using luciferase reporters. The 3′-untranslated region (3′-UTR) of the FUT4-mRNA, which contains a predicted target site for miR-493-5p, was cloned into the GP-miRGLO plasmid. In addition, 3 nucleotides within the predicted miR-493-5p target site were mutated in the GP-miRGLO-FUT4-3′-UTR mutant plasmid. When the 3′-UTR of the FUT4 mRNA was involved in the luciferase transcript, forced miR-493-5p expression decreased luciferase activity by 35% (Fig. 5A), whereas, miR-493-5p mimics had no effect on the luciferase activity of the reporters containing a mutant FUT4 3′-UTR. The results indicated that miR-493-5p targeted the 3′-UTR of the FUT4 mRNA and repressed its expression.

To determine whether FUT4 expression is indeed regulated by miR-493-5p in vitro, the MDA-MB-231 cells were transfected with miR-493-5p agomir, while the MCF-7 cells were transfected with the miR-493-5p antagomir. As expected, miR-493-5p agomir-treated cells showed higher expression of miR-493-5p, and miR-493-5p antagomir inhibited miR-493-5p expression (Fig. 5B and E). The MDA-MB-231 cell line with miR-493-5p expression showed a significant attenuation of FUT4 expression at both the mRNA and protein levels in vitro. Moreover, inhibition of miR-493-5p in MCF-7 cells led to the opposite changes in FUT4 expression (Fig. 5C, D, F and G).

Since the expression levels of miR-493-5p were lower in the MDA-MB-231 cells and breast cancer tissues when compared with the levels in MCF-7 cells and matched adjacent tissues, we examined how alterations in miR-493-5p influence metastatic behavior. MCF-7 and MDA-MB-231 cells were transfected with antagomir or agomir of miR-493-5p, respectively. The effects of the antagomir and the agomir on the invasive rate of MDA-MB-231 and MCF-7 cells were studied with Transwell assay (Fig. 6A and B). Forced miRNA-493-5p overexpression resulted in 41% reduction in the invasive potential of the MDA-MB-231 cells compared to the control cells. The invasive potential was increased in the MCF-7 cells transfected with the antagomir. The enhanced expression of FUT4 significantly increased or reversed the invasive capabilities of the MCF-7 cells (Fig. 6F and G).

To ascertain whether miR-493-5p could further affect the tumorigenicity in vivo, MDA-MB-231, MDA-MB-231/NC and MDA-MB-231/agomir cells were injected into nude mice and tumor formation was monitored. The tumor volume in the MDA-MB-231/agomir group was decreased compared to that noted in the MDA-MB-231 and MDA-MB-231/NC groups (Fig. 6C). Downregulation of FUT4 was also observed in the MDA-MB-231/agomir group (Fig. 6D and E).