MDA-MB-231 and MCF7 cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10% foetal calf serum (PAA Laboratories Ltd., Somerset, UK), streptomycin and penicillin. Cells were incubated at 37 °C, 5% CO₂ and 95% humidity.

Breast cancer tissues (n = 114) and normal background tissues (n = 31) were collected immediately after surgery and stored in the deep freezer at −80 °C. Patients were routinely followed clinically after surgery. The median follow-up period was 120 months. The presence of tumour cells in the collected tissues was verified by examination of frozen sections following H & E staining by a consultant pathologist. Clinical details of the patients are outlined in Table 1.

DRG1 was found to be expressed in both MCF7 and MDA-MB-231 breast cancer cell lines at the transcriptional level using conventional primers outlined in Table 2. Hammerhead ribozyme transgenes, specifically targeted to DRG1 transcripts were constructed based on the secondary structure of DRG1 mRNA. Following design and synthesis by Invitrogen (Paisley, UK), the ribozymes were first amplified using the respective oligos (Table 2) using touch down PCR and were cloned into a mammalian pEF6/His TOPO vector (Invitrogen) and transfected into MCF7 and MDA-MB-231 cells, as previously reported [23,24]. MCF7 and MDA-MB-231 cells were also transfected with an empty pEF6 control plasmid. Cells were then subjected to selection with blasticidin (5 µg/mL) and then subsequently maintained in media containing 0.5 µg/mL blasticidin. This process allowed the generation of stably transfected MCF7 and MDA-MB-231 cells containing the ribozyme transgene and expressing reduced DRG1 levels.

MCF7 and MDA-MB-231 cells containing the control pEF6 plasmid were designated as MCF7^pEF6 and MDA-MB-231^pEF6 and those containing the ribozyme transgenes were labelled MCF7^DRG1KD and MDA-MB-231^DRG1KD. Wild type cells without any plasmid and transgene were called MCF7^WT and MDA-MB-231^WT. Suppression of DRG1 expression in MCF7^DRG1KD and MDA-MB-231^DRG1KD cells was verified, in comparison to MCF7^pEF6 and MDA-MB-231^pEF6 and wild type, using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis.

RNA was extracted using total RNA isolation reagent in accordance with the provided protocol (TRI reagent, Sigma, Dorset, UK). Sample RNA was quantified using a spectrophotometer (WPA UV 1101, Biotech Photometer, Cambridge, UK) and standardized to a concentration of 500 ng. This RNA was used as a template to reverse transcribe cDNA using an iScript cDNA synthesis kit (Bio Rad Laboratories, Hemel Hempstead, UK). Following cDNA synthesis, samples were probed using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers to check cDNA quality and confirm uniform sample cDNA levels, together with those specific for DRG1 transcript (full primer details are shown in Table 2). Polymerase chain reaction (PCR) was performed in a T-Cy Thermocycler (Creacon Technologies Ltd., Emmen, The Netherlands) using REDTaq^® ReadyMix™ PCR Reaction Mix (Sigma). The PCR reaction consisted of an initial denaturing step of 94 °C for 5 min, 30 cycles of 94 °C for 40 s, 55 °C for 40 s and 72 °C for 1 min and a final extension step of 72 °C for 10 min before holding at 4 °C. PCR products were separated on an agarose gel, stained with ethidium bromide and visualized under ultraviolet light.

Real-time quantitative PCR was also used to assess DRG1 transcript levels as previously reported [25]. Results are given as number of transcripts/μL based on an internal standard and the results were further normalised using the expression of GAPDH in these samples. The Q-PCR technique used the Amplifluor system (Intergen Inc., New York, NY, USA), Q-PCR Master Mix (ABgene, Surrey, UK) and a universal probe (Uniprimer™, Intergen) to record the fluorescence emitted during the reaction. Conditions for Q-PCR were: an initial 15 min 95 °C period followed by 60 cycles of 95 °C for 15 s, 55 °C for 60 s and 72 °C for 20 s. Full details of primers used are given in Table 2.

Cell pellets were lysed with an SDS lysis buffer for 1 h, followed by removal of insolubles after centrifugation at 13,000 rpm. Cell lysates were later quantified using the Bio-Rad DC Protein Assay kit (Bio-Rad Laboratories). Denatured samples with equal protein concentrations were separated using SDS PAGE gels. Proteins were blotted onto nitrocellulose membrane, following which they were probed for specific proteins following the SNAP ID protocol provided (Millipore, Watford, UK). GAPDH was used as internal control. The protein bands were subsequently visualized using the Supersignal TM West Dura system (Pierce Biotechnology, Rockford, IL, USA). DRG1 antibody, raised in goat (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and GAPDH, raised in mouse (Santa Cruz Biotechnologies), were used for probing at a concentration of 1:100 and 1:200 respectively. Anti-goat and anti-mouse secondary antibodies were obtained from Sigma and used at a 1:350 concentration in line with recommended settings outlined in the SNAP ID protocol.

The effect of DRG1 suppression on MCF7 and MDA-MB-231 cell growth rates was assessed using an in vitro growth assay. Cells were seeded at a density of 3,000 cells per well, in 200 μL of medium, into 96 well plates. Triplicate plates were set up and incubated for overnight, 3-day and 5-day periods before analysis. Following incubation, the plates were fixed in 4% formaldehyde (v/v) and stained with 0.5% (w/v) crystal violet and then treated with 10% acetic acid (v/v), prior to colorimetric detection of cell density by spectrophotometric analysis at 540 nm using a Bio-Tek ELx800 multi-plate reader (Bio-Tek Instruments Inc., Winooski, VT, USA).

This was based on a previously published protocol [25]. In brief, Matrigel™ basement membrane matrix purchased from BD Biosciences (Oxford, UK), was used to pre coat 96-well plates with a 5 µg per well layer. 45,000 cells were added per well in 200 μL of medium. After incubation for 45 min, wells were vigorously washed with BSS to remove unbound cells. Adherent cells were then fixed in 4% formaldehyde (v/v) and stained with 0.5% (w/v) crystal violet. Stained cells were later counted in a number of random fields under a ×20 objective.

The electric cell-substrate impedance sensing (ECIS) system (Applied Biophysics Inc, Troy, NJ, USA) was used to detect and track MCF7 and MDA-MB-231 cell migration as described previously [26,27]. Briefly, 200,000 cells per well in 300 μL of medium containing Hepes buffer were seeded onto ECIS 96W1E arrays and incubated until a confluent monolayer formed over the array electrodes. This monolayer was then wounded electrically to create a simultaneous physical break in the cell monolayer of equal dimensions. Rate of change in impedance, as cells migrated back onto the electrode, was then monitored and measured using the ECIS software provided.

Invasion assays were undertaken using inserts (ThinCert™ containing 8 μm pores, Greiner bio-one, Germany) in 24-well tissue culture plates. Each insert was first coated with 50 μg/insert of Matrigel. 20,000 cells were seeded into each insert. After 72 h, cells that had invaded and migrated through the matrix and adhered to the underside of the insert were fixed and stained with 4% (v/v) formaldehyde and 0.5% (w/v) crystal violet following a method previously reported [28].

Experimental procedures were repeated independently at least three times. In all assays the transfected breast cancer cell lines, containing the DRG1 ribozyme transgenes, were compared with respective pEF6 plasmid controls (cells containing closed pEF6 plasmid only) using a two-sample, two-tailed t-test. The data values given represent the mean value ± SEM, and values of p ≤ 0.05 were considered to be statistically significant.

Levels of DRG1 transcript were analyzed in connection with tumour grade. No significant difference in DRG1 transcript levels between grade 1 (well differentiated), grade 2 (moderately differentiated) and grade 3 (poorly differentiated) tissues was observed (Figure 1A). NPI (Nottingham Prognostic Index) was also used as an indicator to assess the relationship between DRG1 transcript and predicted prognosis. Patients were divided into groups i.e., good, moderate and poor prognosis according to NPI values. No significant difference was observed among these groups (Figure 1B). Moreover, the relationship between DRG1 expression and TNM status was also analyzed. Similarly, no significant differences in DRG1 transcript expression levels were observed between early stage (TNM1) and later stage (TNM2, 3 or 4) tissues (Figure 1C).

Patients were divided in relation to clinical outcome at final follow up into patients who remained disease free, those with metastasis, those with local recurrence and those who died of breast cancer. Patients who were disease-free were found to have significantly higher levels of DRG1 transcripts than those who developed metastasis and those who died of breast cancer (p = 0.036 and 0.0048 respectively, Figure 1D). Additionally, lower levels of DRG1 tended to be associated with a poorer overall patient survival (Figure 1E) and disease free survival (Figure 1F). Patients with low levels of DRG1 expression had a mean survival time of 122.5 months (95% CI 111.2–133.8) compared to 146.6 months (95% CI 133.5–159.7). Similarly, patients with lower expression levels of DRG1 also had a reduced disease free survival [118.2 months (95% CI 106.4–130.0)] compared to those with higher levels of DRG1 [140.1 months (95% CI 123.2–157.0)]. However, neither overall survival nor disease free survival rates for those with low compared to those patients with high levels of DRG1 was found to be statistically significant (p = 0.133 and 0.176 respectively).

DRG1 expression was observed in both the MCF7 and MDA-MB-231 human breast cancer cell lines available (Figure 2A). Ribozyme transgenes, were subsequently designed based on the secondary structure of the DRG1 transcript (Figure 2B), to target DRG1 expression in these cells. Transfection of MCF7 and MDA-MB-231 breast cancer cells with the DRG1 ribozyme transgene successfully knocked down the expression of this molecule (Figure 2C–F). A reduction in DRG1 expression was observed in MCF7^DRG1KD and MDA-MB-231^DRG1KD compared to the respective wild type (MCF7^WT and MDA-MB-231^WT) and plasmid control (MCF7^pEF6 and MDA-MB-231^pEF6) cells (Figure 2C). Quantitative PCR similarly showed successful transcript knock down of DRG1 in MCF7^DRG1KD and MDA-MB-231^DRG1KD compared to the respective control lines (Figure 2E,F). In keeping with the knock down of transcript expression, reduced protein levels were also observed in MCF7^DRG1KD and MDA-MB-231^DRG1KD, compared to the respective controls, using Western blot analysis (Figure 2D).

The knockdown of DRG1 expression had little impact on the growth of the tested breast cancer cells over the 3 and 5 day incubation periods. At both time points no significant differences were observed between MCF7^DRG1KD or MDA-MB-231^DRG1KD and the respective control cells (p > 0.05) (Figure 3A,B).

Interestingly, knockdown of DRG1 seemed to impact on cell-matrix adhesion differently in the two tested breast cancer cell lines. A significant decrease in adhesiveness was observed between MCF7^DRG1KD cells and control MCF7^pEF6 (p = 0.023), while no significant differences were observed between MDA-MB-231^DRG1KD and MDA-MB-231^pEF6 (Figure 3C,D).

MCF7^DRG1KD cells were found to be significantly more invasive, compared to MCF7^pEF6 cells (p = 0.013) in the Matrigel in vitro invasion assay. In contrast to this, once again, DRG1 knockdown in MDA-MB-231 cells seemed to have very little impact on cellular invasiveness and MDA-MB-231^DRG1KD cells showed no significant change in invasiveness compared with control MDA-MB-231^pEF6 cells (Figure 4A,B).

The electric cell substrate impedance sensing (ECIS) system was used to examine the effect of DRG1 knockdown on breast cancer cell migration. Following electrical wounding, MCF7^DRG1KD cells, migrated at a much faster rate to re-cover the electrode compare to MCF7^pEF6 cells as detected by the enhanced rate of change in resistance detected by the ECIS system. In contrast to this, MDA-MB-231^DRG1KD cells showed no significant change compared to control cells (Figure 4C,D).