br Imaging and Segmentation br miR
Imaging and Segmentation
miR-182 ISH was imaged on the Vectra Automated Mul-tispectral Imaging System (PerkinElmer) at the Research Histology and Tissue Imaging Core at UIC. Cores were imaged at 20 magnification and tiled. Images were un-mixed using a spectral library for Vector Red signal created using Nuance software version 3.0.2 (PerkinElmer) with inForm Advanced Image Analysis software version 2.4.1 (PerkinElmer). For the Outcome TMA, cores that could be unambiguously identified and that contained ISH signal were segmented by a pathologist (A.M.A.) using an adja-cent H&E reference slide. Epithelium of adenocarcinoma, benign glands, and high-grade prostatic intraepithelial neoplasia (PIN) were manually selected in each core for quantitation of Vector Red fluorescence. Representative areas were circled, and, in cores with more than a single type of epithelium of interest (eg, adenocarcinoma and benign prostate glands), equivalent amounts of each cell type were selected. Adenocarcinoma was identified ac-cording to architectural and cytologic parameters: increased nuclear size, decreased nucleocytoplasmic ratio or promi-nent nucleoli, crowded acini, fused glands, cribriform masses, solid sheets, and/or single infiltrating cells. High-grade PIN was identified by nuclear enlargement, prominent nucleoli, and epithelial cell crowding with visible basal BMS-791325 and absence of diagnostic criteria for intraductal carcinoma of the prostate.28 Glands with simple atrophy without inflammation were selected as benign epithelium. All other benign lesions, including histologic variants of atrophy, basal cell hyperplasia, prostatitis, and atypical adenomatous hyperplasia, were excluded. For the Murphy TMA, an adjacent H&E slide was used to select representative benign epithelium, excluding atrophy and PIN from benign cores, and representative adenocarcinoma from tumor cores. Benign epithelium cores were reviewed and approved by a pathologist after segmentation.
Laser-Capture Microdissection and miR-182 Quantification by RT-qPCR
LCM of benign prostate epithelium from frozen prostatectomy tissue and RNA isolation was performed previously.27 Briefly, cDNA was synthesized from 60 ng of LCM-collected
RNA with Universal RT miRNA reagents (Exiqon). qPCR was run in triplicate for miR-182 using the miRCURY LNA miRNA PCR System (Exiqon) and three housekeeping genes (RNU44, RNU48, and RNU66) using SYBR green (BioRad, Hercules, CA) and QuantStudio 6 (Thermo Fisher) with the following settings: 95 C for 10 minutes ( 1), 95 C for 15 seconds ( 50), and 60 C for 1 minute ( 50). For quality control, patients with low housekeeper correlation were excluded from subsequent analysis (Supplemental Figure S1A). miR-182 relative quantity was calculated by the DDCt method, using the mean Ct of the housekeeping genes for normalization.
Correlation of miR-182 Expression with Gene Expression in LCM Benign Epithelium
RNA from 13 African American and 13 patients of European ancestry were analyzed using GeneChip 1.0 Human Gene ST arrays (Affymetrix, Santa Clara, CA) and previously reported in Richards et al27 (Gene Expression Omnibus; https://www. ncbi.nlm.nih.gov/geo; accession number GSE91037). Expression data were normalized using Robust Multiarray Average method in the R package Affy.29 Data quality was evaluated using affyPLM,30 and three arrays (GSM2420027, GSM2420029, and GSM2420033) were excluded from this study based on normalized unscaled SEM plots (Supplemental Figure S1B). Spearman’s r and P values were calculated using the relative quantity of miR-182 expression and the log2 (Robust Multiarray Average) expression values of each probe set for 21 patients using the cor.test function, specifying Spearman correlation, in the stats package of R version 3.3.331 (Supplemental Table S1).
Validation of Predicted miR-182 Targets
Primary prostate epithelial cells were isolated from dei-dentified radical prostatectomy tissue specimens as previ-ously described under UIC Office for the Protection of Research Subjectseapproved Institutional Review Board 2011-1138.14 Cells were grown in PrEGM media (Lonza, Basel, Switzerland) and transfected with miR-182 pre-miRNAs (Thermo Fisher) or mock transfected using siPORT NeoFX (Life Technologies, Carlsbad, CA). After 24 hours, RNA was Trizol (Invitrogen) extracted. cDNAs were made using High-Capacity cDNA RT kit (Invitrogen). Primers are listed in Table 1. qPCR was run using SYBR green and QuantStudio 6 (Thermo Fisher). PCR settings were as follows: 95 C for 10 minutes ( 1), 95 C for 15 seconds, 58 C for 30 seconds, and 72 C for 30 seconds ( 40). Relative quantity was calculated by the DDCt method, using b2-microglobulin for normalization.
Gene-Set Enrichment Analysis
Gene Set Enrichment Analysis version 3.0 software is from the Broad Institute.32 The correlation coefficients for