Comparative microRNA profiling of sporadic and BRCA1 associated basal-like breast cancers

Samples for miRNA analysis

Forty-four primary grade III breast cancer (11 BRCA1 basal, 16 sporadic basal, 17
luminal) and 13 normal breast FFPE (formalin fixed, paraffin embedded) specimens were
collected for the study. Definitions of intrinsic subtypes were based on ER, HER2
in situ hybridization, EGFR and CK5/6 staining, as per Nielson et al. 3]: Basal cancers (ER negative, HER2 negative, CK5/6 and/or EGFR positive), Luminal
cancers (ER positive, HER2 negative). Basal cancers from patients with documented
BRCA1 mutations were sourced from kConFab (www.kconfab.org), whereas normal breast tissue, sporadic basal and luminal cancers were collected
from the Department of Pathology, Peter MacCallum Cancer Centre and the Victorian
Cancer Biobank. Patients with sporadic basal cancers did not have a significant family
history as defined by National Cancer Institute guidelines for BRCA1/BRCA2 mutation
testing (www.cancer.gov). The clinico-pathological characteristics of the patients included in the study
are listed in Additional file 1: Table S1. The study has ethics approval (Peter MacCallum Cancer Centre 09/36). For
patients with sporadic cancers, due to the use of archival FFPE tissue, written informed
consent was not required by the ethics committee. For BRCA1 patients, written informed
consent was obtained as per kConFab (Kathleen Cuningham Foundation Consortium for
research into Familial Breast cancer) biobank guidelines (www.kconfab.org). Three basal (HS578T, MDA-MB-231, MDA-MB-468, all with wild-type BRCA1) and two
luminal (MDA-MB-453, MCF-7) breast cancer cell lines were also included in the study.

RNA extraction

For primary tumours and normal breast tissue, 10 ?m thick sections were cut from FFPE
tissue blocks. The sections were dewaxed in xylene, placed through 100 % alcohol and
allowed to dry. The samples were needle microdissected to ensure the proportion of
tumour (or normal epithelium) was greater than 80 %, prior to placement into lysis
buffer (Agencourt Formapure kit, Beckman Coulter, Beverly, MA, USA). Tissue was digested
as per kit protocol (incubate at 70 ° C for 1 h, then add 20 ?l of Proteinase K and
incubate at 55 ° C for 1 h). Total RNA was extracted via a standard TRIZOL(Sigma)/chloroform
protocol. For cell lines, total RNA was extracted using the total RNA protocol from
the mirVana miRNA Isolation Kit (Ambion, TX, USA). All samples underwent DNase treatment
with the Ambion DNA-free kit (Ambion, TX, USA).

miRNA array

For each sample, 250 ng of total RNA was labelled and hybridized on Human v2 MicroRNA
Expression BeadChips (Illumina, San Diego, CA, USA), according to the manufacturers
recommendations (Illumina MicroRNA Expression Profiling Assay Guide). The layout of
samples across the beadchips is shown in Additional file 1: Table S2. Sixty-nine samples (44 tumour, 13 normal, 7 controls and 5 cell lines)
were hybridised on six beadchips across two separate runs: Run 1 (1 beadchip, 11 samples)
and Run 2 (5 beadchips, 58 samples). The sample groups were randomised across the
beadchips and also based on position within the beadchip. Controls were included for
comparisons between the six beadchips and also between the two separate runs. The
correlation of miRNA from control samples across the beadchips are outlined in Additional
file 2: Figure S1.

The BeadChips were scanned with the Illumina iScan Reader. Data were imported into
GenomeStudio (Illumina), from which raw data with background subtraction were exported
to PARTEK Genomics Suite (St. Louis, Missouri, USA) for further analysis. Probes with
a maximum intensity value of less than 150 units across all samples were excluded.
Of the 1145 probes present on the array, 1037 were used for subsequent analyses. Raw
probe intensities were shifted, such that the minimum probe intensity for each sample
was equal to 1. All values were transformed by taking logs (base 2), followed by quantile
normalisation 18]. Probe mapping for Illumina MicroRNA Expression v2 BeadChips was based on miRBase
v.12.0.

Differential expression between groups was assessed using ANOVA, with inclusion of
the Beadchip number as an independent variable to control for variations between Beadchips.
A p-value of??0.05, after Benjamini-Hochberg adjustment for multiple tests, was regarded
as significant.

For each miRNA, the expression profiles were standardised to a mean of zero and a
standard deviation of 1 prior to unsupervised hierarchical clustering. Clustering
was performed using average linkage and Pearson correlation 12]. The full array data is available in GEO (Accession number: GSE61438).

microRNA real time RT-PCR

Expression of microRNA (miRNA)s hsa-miR-374b, ?190b, ?198, ?892a, ?130b*, ?218, ?590-3p
and ?149 was assayed using real time RT-PCR. cDNA was reversed transcribed from total
RNA samples using TaqMan MicroRNA assays and the TaqMan MicroRNA reverse transcription
kit (Applied Biosystems). The cDNA was amplified using TaqMan microRNA Assay primers
and the TaqMan Universal PCR Mastermix, according to the manufacturer’s instructions
on the Roche Lightcycler 480. The relative miRNA expression levels were calculated
by normalisation with RNU6B expression 19] using the second derivative (Cp) method 20]. Comparisons between groups were made using the un-paired t-test and correlations with array data were investigated using Pearson correlation
on GraphPad Prism 5 (La Jolla, CA, USA).

Prediction of miRNA targets

Predicted targets of miRNAs were identified via a union search of the two target prediction
algorithms miRBase and TargetScan 5.1. Analysis of target protein expression by pSILAC
(pulsed stable isotope labelling by amino acids in cell culture) suggests the specificity
of these two algorithms are 44 and 61 % respectively 21]. Hence to improve specificity, only genes that are the predicted targets of three
or more miRNAs differentially expressed between tumour groups was reported.

Ago2 immunoprecipitation studies by Karginov et al. have shown that approximately
20 % of mRNA targets undergo miRNA-induced cleavage. For the remainder of the targets
(80 %), protein translation is suppressed without changes in mRNA levels 21], 22]. To identify the subset that undergoes miRNA-mediated cleavage, predicted targets
derived from above were cross referenced with gene expression array data.

Gene expression array

Gene expression data for 14 BRCA1 and 10 non-BRCA1 (5 BRCA2 and 5 BRCAX) basal cancers
were derived from a cohort previously described by Waddell et al. (Data available
on GEO, accession number GSE19177) 23]. RNA was extracted (Qiagen, Doncaster, VIC) from fresh frozen tissue and gene expression
profiling was performed as per the manufacturer’s guidelines using 450 ng total RNA
and Illumina Human-6 version 2 BeadChips containing 46,000 probes (Illumina Inc.,
San Diego, CA). Raw data were imported from Illumina Beadstudio v3.2 to PARTEK for
further processing. Data were normalized with quantile normalisation, then filtered
using an Illumina detection score of??0.95 in at least one sample, which yielded
24,004 probes that were used in further analyses.

Immunohistochemistry for miRNA targets

Tissue microarrays (TMAs) with single 1 mm cores were constructed. Cyclin D1, FOXP1,
FIH-1, pan-ER?, NRP1 and CD99 immunohistochemistry was performed on TMAs constructed
from a cohort of 35 BRCA1 basal cancers from kConFab, and 52 sporadic basal cancers
from the Instituti Ospitalieri di Cremona, Italy. Selection of antibodies was based
on their previously described associations with BRCA1 status, wherever possible 24]–28]. Immunohistochemistry for cyclin D1, FOXP1, NRP1 and CD99 was repeated on a second
validation cohort composed of 82 BRCA1 basal cancers from kConFab, and 65 sporadic
basal cancers from the Peter MacCallum Cancer Centre, Melbourne. TMA sections were
cut from each block at 4 ?m thick intervals, dewaxed, and placed through graded alcohol
and placed into water. The antibody clones used and their titrations are listed in
Additional file 1: Table S3. Antigen retrieval, incubation and visualisation for FIH and pan-ER? were
performed as per previous published studies 27], 29]. For NRP1 antigen retrieval was performed in a pressure cooker using high pH EnVision
FLEX Target Retrieval Solution (Dako, Glostrup, Denmark) for 2 min. Antigen-antibody
complex was detected using Envision FLEX system. FOXP1 and cyclin D1 staining was
performed on the Ventana Benchmark® ULTRA system. Antigen retrieval was performed
using Ventana ULTRA Cell Conditioner 1 and visualized with Ventana Ultraview Universal
DAB. The intensity of staining was scored as negative?=?0; weak staining?=?1; moderate
staining?=?2; or strong staining?=?3. The percentage of tumour cells stained in the
given core scored as: 0 %?=?0; 1–10 %?=?1; 11–50 %?=?2; 51–80 %?=?3; 81–100 %?=?4.
The scores for both staining intensity and the percentage of positive tumour cells
were added together to give a maximum score of 7. Comparisons between groups were
based on a chi square (based on presence or absence of staining) and Mann-Whitney
U tests (based on scores out of 7) performed on SPSS 16.0 (SPSS, IL, USA).