Growth and maintenance of cell lines
The breast cancer cell line MDA–MB–361 was cultured in DMEM (Dulbecco Modified Eagles medium) with 20% FCS, (Invitrogen, Carlsbad, CA) and T47D was maintained in RPMI 1640 (Roswell Park Memorial Institute medium) supplemented with 10% FCS and human insulin (10 μg/ml). The cell cultures were maintained in a water humidified 37°C incubator with 5% CO2.
Ionizing radiation treatment
Irradiation of cell cultures containing 1 × 106 log phase cells was performed with a Cs-137 irradiator (HWM D-2000, Siemens, Germany) at a dose rate of 0.95 Gy/min. Doses of 2.5 Gy; 5.0 Gy or 7.5 Gy were administered at room temperature and control cells were sham irradiated. The exposed and sham irradiated cells were subsequently incubated at 37°C and harvested after indicated time points for RNA and protein isolation. The experiment was repeated for each dose in triplicate.
Lentivirus production and infection of breast cancer cell lines
Replication-defective lentiviral particles were produced by transient co-transfection of HEK293T cells in a 10 cm petri dish with 16 μg, 8 μg and 4 μg of packaging plasmids pMDLg/pRRE, pRSV. Rev and pMD2.G (a kind gift from D. Trono, École polytechnique fédérale de Lausanne) and 8 μg of lentiviral transduction vector pGreenPuro (pGP; System Biosciences, California) using Lipofectamine 2000 (Life Technologies, California) according to the manufacturer’s instructions. The pGP vector (named EV – empty virus in results section) was used as the backbone for miR-21 overexpression and miR-21 downregulation (anti-miR-21) by specific miRNA oligo cloning (pmiRZIP-21 - Cat. Nr. MZIP21-PA-1-GVO-SB; Biocat, Heidelberg, Germany).
The virus particles were harvested 48 hours after transfection, cleared and concentrated as previously described
. According to virus titer determination virus productions ranged between 108 and 109 TU/ml (TU - Transduction Units). Viral infection of breast cancer cells was performed using protocols previously described
. Briefly, 2 × 105 cells per well were infected with 4 × 105 TU/ml (defined as 2 MOI – multiplicity of infection) and three days after infection GFP expression was monitored. After infection 5 × 105 cells were irradiated for indicated time points. Microscopic analysis was done 48 hours post irradiation (HBO 50/AC and AxioCam MRC, Carl Zeiss AG, Germany).
RNA isolation for miRNA expression analysis
Paraffin-embedded tissue was microdissected with a sterile needle from 5 μm thick sections using a stereo microscope (Stemi 2000, Zeiss, Germany). A consecutive H&E-stained section was used for guidance. Tumour cell material (containing at least >80% tumour cells) was collected from all cases. Additionally, histologically normal ductal epithelium material was collected from five cases as control tissue. Total RNA was isolated from microdissected tissues as previously described
. After digestion in lysis buffer and 500 μg/ml proteinase K the RNA was purified by phenol/chloroform extraction, ethanol precipitated, and dissolved in 20 μl RNase-free water. Five microlitres (100 ng) of RNA were reverse-transcribed using MultiScribeTM reverse transcriptase (Applied Biosystems; Foster City, CA, USA)
. Further processing and evaluation of the results was performed according to the manufacturer’s instructions.
Total RNA was isolated from each of the breast cancer cell lines (MDA-MB-361 and T47D) after irradiation. Cells were pelleted by centrifugation at 1500 rpm for 5 min, and washed with 1 ml Dulbecco’s phosphate-buffered saline (PBS) without MgCl2 and CaCl2 (Invitrogen, Carlsbad, CA, USA). Small RNAs (<200 nucleotides) were isolated from the cells using the mirVana™ miRNA isolation kit (Applied Biosystems; Foster City, CA, USA) following the protocol for total RNA isolation. The quantity and quality of the total RNA and miRNA was measured with the Nanodrop spectrophotometer (PeqLab Biotechnology; Germany) and by running 2% agarose gels stained with ethidium bromide, respectively.
TaqMan-miRNA assays and data analysis
A specific single TaqMan – miRNA assay (Applied Biosystems, Forster City, CA, USA) was used for miR-21 expression analysis (Cat.Nr. 4427975; Assay ID 000397) in total RNA isolations from FFPE samples and from cells treated with irradiation. Quantitative PCR was performed on StepOnePlus Detection System (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. The relative expression values of specific miRNA were calculated by using the 2–ΔΔCT method
 normalized to the control miRNA (RNU43 and RNU44 - Cat.Nr. 4427975; Assay ID 001094 and 001095) and to the FFPE control or non-irradiated sample. All reactions were performed at least twice in duplicate.
Cell Proliferation and survival
Cell proliferation and viability was determined with a colorimetric cell proliferation WST1 kit (Roche, Manheim, Germany). Twenty-four hours before irradiation, 1000 to 2000 cells per well were seeded into 24-well plates. Three days after irradiation, 200 μl fresh growth medium and 20 μl WST1 labeling reagent were added and the cells were incubated for 2 hours in a 37°C incubator with 5% CO2. After incubation the absorbance was determined at 450 nm with reference length at 650 nm using a spectrophotometer plate reader (TECAN, Switzerland). For the measurement of clonogenic survival, cells were seeded in range of densities (500–2000 cells per plate) and 24 h later irradiation was performed. After 10–14 days, the colony formation capacity was assayed after ethanol fixation and Giemsa staining.
Cell cycle and subG1 fraction analysis
DNA staining of isolated nuclei for cell cycle analysis was performed using a modification of the method of Nüsse et al.,
. At each indicated time, the treated cells were trypsinized and collected by centrifugation at 300 g for 5 min, and the supernatant was carefully removed. The cell pellet was gently resuspended in 500 μl of a solution containing 10 mM NaCl, 4 mM Na-citrate, 10 μg/ml RNase, 0.3% Nonidet P-40, and 50 μg/ml propidiumiodide (PI). The cell suspensions were incubated for 60 min at room temperature followed by the addition of 500 μl of solution containing 70 mM citric acid, 250 mM sucrose and 50 μg/ml PI. The cell suspensions were mixed and stored at 4°C before flow cytometry. Cell cycle distributions were analyzed on a FACScan LSR II (Becton- Dickinson) (excitation wavelength: 488 nm; emission wavelength: 610 nm, LSR II, Becton Dickinson/FACS DIVA Software). Cells with a DNA content less than that of cells in the G1 phase of the cell cycle (<2n) were assigned to the subG1 fraction and were considered to be apoptotic.
Patients and tumour samples
Formalin-fixed and paraffin-embedded (FFPE) archival material, obtained from 86 patients with invasive ductal breast carcinomas (IDC), was used for miRNA analysis. Forty-nine tumours were lymph node negative and 57 tumours were small in size (≤2 cm). Nine of the tumours were histological grade 1, 56 were grade 2, and 23 were grade 3
[29, 30]. The patients age ranged from 15 to 84 years (median 66 years). All patients were surgically treated, and no patient received preoperative adjuvant chemotherapy treatment. Postoperative 29 patients received radiation therapy treatment and 4 patients received Novaldex with radiation therapy. Detailed long-term clinical follow-up was available for all patients with a median follow-up period of 113 months (min. 5 months, max. 468 months). Forty patients relapsed with distant metastases within the total follow-up period. Ethical approval for the study was obtained from the Ethics Committee of the Medical Faculty of the Technical University of Munich.
Correlation between histopathological markers and miRNA expression was examined by Spearman's rank correlation test. For univariate survival analysis Kaplan-Meier curves were calculated from 86 patients, and differences between strata were tested with the log-rank Chi-Square value. Results obtained in the in vitro experiments were tested using one- or two-way ANOVA and GraphPad Prism. In all analysis statistical significance was considered at the p <0.05 levels.