The MCF-7 human breast cancer cell line was purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, Shanghai Institute of Cell Biology, Chinese Academy of Sciences, (Shanghai, China) and was cultured in Dulbecco’s modified Eagle’s medium (Gibco Life Technologies, Gaithersburg, MD, USA), supplemented with 10% fetal bovine serum (Gibco Life Technologies), 100 U/ml penicillin (Sigma-Aldrich, St. Louis, MO, USA) and 0.1 mg/ml streptomycin (Sigma-Aldrich), in a humidified atmosphere of 5% CO₂ and 37°C.

A full-length open reading frame of the human GRIM-19 gene was cloned from the MCF-7 cells using polymerase chain reaction (PCR). The primers (Takara Biotechnology Co., Inc., Dalian, China) for GRIM-19 cloning were as follows: Sense, 5′-GAGAATTCATGGCGGCGTCAAAGG-3′ (EcoRI) and antisense 5′-GACTCGAGCAGGGCCTACGTGTACCACAT-3′ (XhoI). The cDNA of GRIM-19 was cloned into the XhoI and EcoRI sites of the pcDNA3.1 mammalian expression vector (Invitrogen Life Technologies, Carlsbad, CA, USA) and was termed pGRIM-19. The successful construction of the recombinant plasmid, pGRIM-19, was confirmed by DNA sequencing (BIG, Guangzhou, China). pGRIM-19 was transfected into the MCF-7 cells using Lipofectamine 2000 (Invitrogen Life Technologies), according to the manufacturer’s instructions. Following 72 h culture at 37°C, the cells were harvested to determine the mRNA and protein expression levels of GRIM19 using RT-qPCR and western blotting, respectively.

The mRNA expression levels of signal transducer and activator of transcription 3 (STAT3) and GRIM-19 were assessed using RT-qPCR. The cells, which were transfected with the pGRIM-19 plasmids were collected by centrifugation at 1,000 × g for 5 min at room temperature 72 h after transfection. The total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies). The first strand cDNA was reverse transcribed with 2 μg total RNA, using a Takara Reverse Transcription kit (Takara Biotechnology Co., Inc.) and oligo (dT) 15 primers (Takara Biotechnology Co., Inc.). Amplification of GRIM-19 was performed with 1 cycle at 95°C for 5 min, and 30 cycles of 95°C for 15 sec, 55°C for 30 sec and 72°C for 1 min, with a final extension for 5 min The primers used for STAT3 and GAPDH were as follows: STAT3, sense 5′-GAGTCAGGCACTGTGGG-3′ and antisense 5′-CGGTCGGTTTCTGCCTGTA-3′ and GAPDH, sense 5′-CCTTCATTGACCTCAACTA-3′ and antisense 5′-GGAAGGCCATGCCAGTGAGC-3′. GAPDH was used as a control. The qPCR reactions were performed on an ABI 7900 Fast system (Applied Biosystems, Foster City, CA, USA) as follows: Initial denaturation at 94°C for 5 min, followed by 30 cycles of amplification (94°C for 30 sec, 58°C for 45 sec and 72°C for 90 sec) and a final extension of 10 min at 72°C. The reaction products were analyzed using 1.5% standard agarose gel electrophoresis (Sigma-Aldrich) and visualized using a GIS Gelatumimaging system (Tanon, Shanghai, China).

The culture supernatants were collected by centrifugation at 1,000 × g for 5 min at room temperature and concentrated ~20-fold using a spin-concentrator (Millipore, Bedford, MA, USA) 72 h after transfection. The proteins from the cell supernatants and the lysates were measured using a bicinchoninic acid (Sigma-Aldrich) method. Equal quantities of protein (30 μg) were loaded into each lane of 10–15% SDS-PAGE gels (Sigma-Aldrich) and subsequently transferred onto polyvinylidene difluoride membranes (Sigma-Aldrich). The membranes were blocked with 5% non-fat milk to inhibit non-specific binding, and the membranes were then incubated overnight at 4°C with the following antibodies: Rabbit polyclonal anti-human Survivin (1:5,000; cat. no. 2803S; Cell Signaling Technology, Inc., Danvers, MA, USA), mouse monoclonal anti-human B-cell lymphoma (BCL)-2 (1:3,000; cat. no. sc-7382; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit polyclonal anti-human matrix metalloproteinase (MMP)-2 (1:1,000; cat. no. 4022S; Cell Signaling Technology, Inc.), mouse monoclonal anti-human MMP-9 (1:2,000; cat. no. sc-12759; Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-human urokinase-type plasminogen activator (u-PA; 1:2,000; cat. no. sc-14019; Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-human GRIM-19 (1:1,000; cat. no. sc-365987; Santa Cruz Biotechnology, Inc.) and mouse monoclonal anti-human Stat3 (1:2,000; cat. no. sc-8019; Santa Cruz Biotechnology, Inc.). Mouse monoclonal anti-human GAPDH (1:10,000; cat. no. sc-365062; Santa Cruz Biotechnology, Inc.) was used as a loading control. The membranes were then incubated with polyclonal goat anti-mouse horseradish peroxidase-conjugated immunogloblin G (1:10,000; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) or polyclonal goat anti-rabbit horseradish peroxidase-conjugated immunoglonulin G (1:10,000; cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature and the protein bands were visualized using a supersignal enhanced chemiluminescence detection kit (Pierce, Rockford, IL, USA).

Cell proliferation was assessed using an MTT cell proliferation kit (Roche Applied Science, Indianapolis, IN, USA), according to the manufacturer’s instructions. Briefly, the cells (2×10³/well) were seeded into a 96-well plate and incubated for 2 h at 37°C. Cell growth was measured using color generation (MTT reduction) by adding 20 μl MTT (5 mg/ml) to the culture medium. The cells were harvested and incubated with 100 μl dimethyl sulfoxide (Sigma-Aldrich) for 30 min, and absorbance was measured using an enzyme-linked immunosorbent assay (ELISA) multi-well spectrophotometer (SpectraMax M3; Molecular Devices Corp., Sunnyvale, CA, USA) at a 490 nm test wavelength. All assays were performed in triplicate and data are expressed as the mean ± standard deviation.

The cells were seeded at a density of 1,000 cells/well in 24-well tissue culture plates. The plates were incubated for 2 weeks in a humidified incubator at 37°C. After 3 weeks, the colonies were stained using 0.05% crystal violet (Sigma-Aldrich), containing 50% methanol (Sigma-Aldrich). The colonies were counted in between four and five randomly selected fields for each of the duplicate samples using a microscope (IX51; Olympus, Tokyo, Japan) at magnification, ×100.

For the analysis of apoptosis, the MCF-7 cells were collected by centrifugation at 1,000 × g for 5 min at room temperature 48 h after transfection, diluted to a concentration of 1×10⁶ cells/ml and washed three times with ice cold phosphate buffered saline (PBS; Sigma-Aldrich). The cells were then incubated with phycoerythrin (PE) annexin-V and 7AAD using a PE Annexin V Apoptosis Detection kit I (BD Pharmingen, San Diago, CA, USA), according to the manufacturer’s instructions. The cells were then analyzed using fluorescence-activated cell sorting (FACS Calibur; BD Pharmingen, San Diago, CA, USA). The experiments were performed in triplicate. In addition, the expression levels of survivin and Bcl-2 were also assessed using western blot analysis as an additional indicator of apoptosis.

A wound-healing assay was performed to assess the effect of GRIM-19 on cell migration. Briefly, the cells (2×10⁴) were cultured to confluency and transfected with the pGRIM-19 plasmid (1 μg) for 4 h at 37°C to arrest cell proliferation. A wound track was made using a 200 μl pipette tip and the medium and any cell debris were removed. The plates were washed with PBS and the cells were grown in fresh medium for a further 24 h. The cells invading the wound line were observed under an inverted phase-contrast microscope (Leica DMR; Leica, Wetzlar, Germany).

Cell invasion was measured using a Matrigel-coated film insert (8 μm pore size) in 24-well invasion chambers (BD Biosciences). The cells (5×10⁴), which were suspended in 200 μl FBS-free DMEM, were added to the upper compartment of the invasion chamber and 500 μl complete media was added to the lower chamber. Following incubation for 24 h, the cells on the upper side of the filter were removed and the cells in the lower surface of the filter were stained with hematoxylin and eosin (Sigma-Aldrich). The cell number was counted using a microscope (Olympus, Tokyo, Japan) at a magnification of ×200. The mean values were calculated by averaging the total numbers of cells from ten randomly selected microscopic fields. The assay was performed in triplicate.

VEGF production was determined using a competitive ELISA. Briefly, the MCF7 cells (2×10⁴) were transfected with the pGRIM-19 plasmid in 12-well plates for 24 h at 37°C. The culture media was subsequently centrifuged to remove the cell debris. The cell-free culture media was collected by centrifugation at 1,000 × g for 5 min at room temperature at indicated time-points and the levels of VEGF were measured using a Human VEGF ELISA kit (Yanyu, Shanghai, China), according to the manufacturer’s instructions.

All experiments were performed at least three times independently and the data are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS software (version 19.0; SPSS Inc., Chicago, IL, USA) and GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA, USA) for Windows^®.

GRIM-19 was amplified using PCR and subcloned into the pcDNA3.1 vector. The resulting plasmid, pGRIM-19, was them confirmed by sequence analysis. The results of sequence alignment demonstrated that the GRIM19 gene shared a sequence homology of 100% with the GRIM-19 published in GeneBank (no. AF155662). The pGRIM-19 or pcDNA3.1 blank plasmid were transfected into the MCF7 cells, and the mRNA and protein expression levels of GRIM-19 were determined using RT-qPCR and western blot analyses, respectively. The results of the RT-qPCR revealed that the expression of GRIM-19 was significantly increased in the MCF-7 cells transfected with the pGRIM-19 plasmid, compared with the untreated cells and the cells transfected with the blank vector (P<0.05; Fig. 1A and B). The protein expression of GRIM-19 was also significantly upregulated following transfection with the pGRIM-19 plasmid (P<0.05; Fig. 1C and D).

To evaluate the association between GRIM-19 and its target gene, STAT3, in breast cancer cells, the present study examined the effects of upregulated GRIM-19 on the expression of STAT3 in breast cancer cells. As shown in Fig. 2A, increased levels of GRIM-19 in the MCF-7 cells resulted in a marked decrease in the mRNA expression of STAT3 at 72 h after transfection (Fig. 2A). Similar results were confirmed in the western blot analysis (Fig. 2B).

To investigate whether the upregulation of GRIM-19 affects cell proliferation, an MTT assay was performed. The results revealed that increased levels of GRIM-19 in the MCF-7 cells resulted in a notable decrease in cell proliferation (P<0.01; Fig. 3A).

In addition, the effects of the upregulation of GRIM-19 on breast cancer cell colony formation ability were assessed. As shown in Fig. 3B, overexpression of GRIM-19 in the MCF-7 cells reduced the colony number, compared with those observed in the blank vector or control groups (P<0.05).

To investigate whether upregulation of GRIM-19 induced cell apoptosis, the apoptotic rate of the cells was assessed 48 h after treatment with pGRIM-19. Significantly higher levels of apoptosis were observed in the MCF-7 cells transfected with the pGRIM-19 plasmid, compared with the blank vector or control groups (P<0.05; Fig. 4A).

To determine the potential mechanism of GRIM19-induced cell apoptosis in vitro, the protein expression levels of survivin and Bcl-2 were determined using western blot analysis The results demonstrated that increased expression of GRIM-19 in the MCF-7 cells resulted in a notable decrease in the protein expression levels of survivin and Bcl-2, compared with the blank vector or control groups (P<0.05; Fig. 4B).

To ascertain the inhibitory effect of the upregulation of GRIM-19 on breast cancer cell motility in vitro, a wound-healing assay was performed. As shown in Fig. 5A and B, the MCF-7 cells transfected with pGRIM-19 migrated significantly less than the untreated cells and the cells transfected with the blank vector (P<0.01)

The ability of upregulated GRIM-19 to reduce the invasiveness of breast cancer cells was further investigated using a Transwell system assay. The invasion of the cells was also significantly decreased in the pGRIM-19 treatment group, compared with the control and the blank vector groups (P<0.01; Fig. 5C and D).

The observation that GRIM-19 exerts an inhibitory effect on the migration and invasion of breast cancer cells, prompted examination of its effects on the expression levels of u-PA, MMP-2, MMP-9 and VEGF in breast cancer cells. As shown in Fig. 6A, ELISA analysis revealed that the level of VEGF excretion in the supernatant from the pGRIM-19-transfected cells was significantly decreased (P<0.01), compared with the blank vector and control groups. Western blot analysis revealed a marked decrease in the protein expression levels of MMP-2, MMP-9 and u-PA in the supernatant and the total extracts from the pGRIM-19-transfected cells, compared with the blank vector and control groups (Fig. 6B). These findings suggested that the inhibitory effect of GRIM-19 on the invasion and metastasis of breast cancer was, at least partially, mediated by the down-regulation of u-PA, MMP-9, MMP-2 and VEGF, which may contribute to degradation of the extracellular matrix (ETM).