Fluorescence based immunohistochemical staining was performed as previously described (12-16). PTEN was diluted 1:100 (Cell Signaling Technologies CST138G6). c-Met antibody Met4 from George Vande Woude diluted 1:5,000 (17). ERα was diluted 1:50 (Dako 1D5). ERβ1 was diluted 1:500 (Thermo Scientific PPG5/10). Her4 (Biotechnology, Santa Cruz, CA, USA) was diluted 1:2500. Her3 (Cell Signaling Technology, Beverly, MA, USA) diluted 1:100. E2F4 (Novus SPM179, Littleton, CO, USA) was diluted 1:100. BCL2 (Dako clone 124) diluted 1:1,000. Zeb1 (Sigma Aldrich HPA027524) diluted 1:500.Quantitative image analysis by AQUA
The AQUA method quantifies fluorescent signal within subcellular compartments as described previously (15). AQUA begins with a series of high resolution monochromatic images for each field of view using the signal from DAPI, Cy3 (cytokeratin), and Cy5 (miRNA). The AQUA algorithm first creates a binarized tumor mask (distinguishing tumor from stroma) based on the cytokeratin signal. Next subcellular compartments within the tumor mask are determined by creating the nuclear compartment (as determine by DAPI positive pixels) and subtracting the nuclear compartment from the tumor mask to create the cytoplasmic compartment. Lastly, AQUA scores for the miRNA or protein of interest are calculated by dividing the signal intensity of CY5 (scored on a scale from 0–255) by the area of the tumor mask or compartment of interest, then normalized to the illumination source intensity.Patient cohorts
Use of human tissue in this study was approved by the Yale institutional IRB, HIC protocol 9500008219 including consent and waived consent. AQUA, Yale tissue microarrays, YTMA-49 (The Yale Breast Cancer Cohort), YTMA-79 (The Yale Lung Cancer Cohort), and microarray construction has been described previously (15,–16, 18). The Yale Breast Cancer Cohort consists of 619 breast cancer patients diagnosed from 1976–1982 at Yale-New Haven Hospital. The Yale Lung Cancer Cohort consists of 197 lung cancer patients who underwent surgery at Yale-New Haven Hospital between 1995–2003. Exclusion of individual histospots as a result of technical failure, attrition of sample, or missing clinical variables results in less than 100% inclusion of all tumor specimens in analyses.Cell culture
Cell culture conditions have been previously described (15). MCF-7 and MDA-MB-231 cells were purchased from ATCC. The cells were transfected with 30 nM anti-miR-221 inhibitor or anti-miRNA negative control (Ambion, Austin, TX, USA) with Lipofectamine RNAi Max (Invitrogen) following manufacturer's instructions and incubated for 48 h before performing ISH on coverslips or preparing RNA extracts.
RNA extracts were prepared using the mirVana miRNAIsolation Kit (Ambion) following the manufacturer's instructions. In performing ISH on coverslips, cells were washed in PBS, fixed with 1% formaldehyde for 10 min (Thermo Scientific), permeabilized with 0.2% Triton X-100 (American Bioanalytical) for 20 min on ice, refixed with 4% formaldehyde for 10 min, peroxidase blocked with 3% H2O2 for 10 min, prehybridized at 40°C for 30 min, hybridized with double DIG 100nM miR-221 or scrambled probe (Exiqon), or 50 nM U6 probe 5′DIG labeled probe (Exiqon) for 1 h, washed with 2xSSC at hybridization temperature twice then once at room temperature, then blocked with 2% BSA-PBS for 30 min, incubated for 1 h with sheep Anti-Digoxigenin-POD, Fab fragments from sheep (Roche Diagnostics) diluted 1:100 in block. Next the miRNA signal is detected with the TSA Cyanine 5 system (Perkin Elmer) and the coverslips were mounted with Prolong Gold-DAPI (Molecular Probes).miRNA qRT-PCR
RNA concentrations were determined by the NanoDrop 2000 (Thermo Scientific), then reverse transcribed using the TaqMan microRNA Reverse Transcription Kit, and real-time PCR using the TaqMan microRNA assay kit for miR-221 (000524) or U6 (1093, Applied Biosystems). RT-PCR was performed using the CFX96 machine (BioRad, Hercules, CA). Reactions were done in triplicate along with no template control reactions, and miR-221 expression was normalized to U6 using the 2-▵▵CT method.Statistical analysis
All values shown are mean ± s.d. unless otherwise stated. Box plots show standard box and whiskers plots where the error bars represent the 90th and 10th percentiles. To test for differences between groups, p-values were calculated by ANOVA analysis. Survival curves were generated by Kaplan-Meier analysis and tested for significance using the Mantel-Cox log rank test. Prognostic significance for miR-221 was determined using the Cox proportional hazards model. All statistical analysis were done using Statview 5.0 (SAS Institute).