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Baumann et al
Table 1 Primers for Quantitative RT-PCR Validation of Predicted miR-182 Gene Targets
CD164 FWD 50 -TTAGCTTTCTCCCGAACGCC-30
RVS 50 -GCAGCTGTTTCGACCTTCAC-30 ELL2 FWD 50 -ATGTGAAGCTCACCGAGACG-30
RVS 50 -TTTGACAAGCCCGTGGAGTC-30 PRKAR1A FWD 50 -ACACCCAGAGAGGGACAGAGAA-30
RVS 50 -GAGCTCACATTCTCGAAGGCT-30 RNF152 FWD 50 -AGACTCGGTGACAGATACAGAAAT-30
RVS 50 -TGCAGGTAATGGCAAGCTCA-30 NR3C1 FWD 50 -GTTGTTTATCTCGGCTGCGG-30
RVS 50 -TCAGTGAATATCAACTCTGGCA-30 B2M FWD 50 -CCTGAATTGCTATGTGTCTGGG-30
RVS 50 -TGATGCTGCTTACATGTCTCGA-30
FWD, forward; RVS, reverse.
genes with multiple probe sets were averaged together. To mimic DETA NONOate data input, the negative correlation co-efficients were multiplied by 1 and input in triplicate as negative correlation samples and the positive correlation coefficients in triplicate as positive correlation samples. Because of the small sample size, gene set permutation was used to calculate statistics. The miR-182-5p targets were predicted from TargetScan version 7.1 with a cumulative-weighted context score < 0.5,33 miRDB with a target score >0.85,34 and TarBase version 8 with a prediction score >0.85.35 Other gene sets were downloaded from MSigDB version 6.1, including Kyoto Encyclopedia of Genes and Genomes
The Cancer Genome Atlas Analysis
The results shown are in part based on data generated by The Cancer Genome Atlas (TCGA) Research Network (http://cancergenome.nih.gov; last accessed May 2, 2018). TCGA prostate adenocarcinoma miRNA sequencing counts data and RNA sequencing counts data (level 3 publicly available data) were downloaded from the National Cancer Institute Genomic Data Commons (https://portal.gdc.cancer.gov; last accessed August 2017) and matched by case identifiation. For small RNA sequencing, counts per million miRNA counts were used as reported, and for RNA sequencing, gene counts were divided by the total number of unique reads per patient 1,000,000 for counts per million. There were 499 tumor cases with both data sets.
Statistical analyses were performed in GraphPad Prism software version 5.04 (GraphPad Software Inc., San Diego, CA) for U-test, Kruskal-Wallis test, and t-test. Statistics were performed in R version 3.3.331 for Spearman correla-tion of microarray gene expression with miR-182 and lo-gistic regression analysis. Interclass correlation coefficients
Figure 1 miR-182 is expressed exclusively in prostate epithelium and higher in areas of prostate cancer. A: Specificity of miR-182 LNA in situ hy-bridization (ISH) probes (red) with nuclear coun-terstain DAPI (blue) assessed in RWPE1 benign prostate cells transfected with control (RWPE1-CTL) or miR-183 clustereexpressing lentivirus (RWPE1-182). Bar graph showing quantification of miR-182 ISH intensity per cell (two-tailed un-paired t-test). B: Representative of no-probe sec-ondary antibody-only control, miR-182 ISH, and U6-positive control developed with Vector Red in benign prostate tissue. DAPI (blue) as nuclear stain. Boxed areas in top row of B are shown in higher magnification in the bottom row. C: miR-182 ISH (red) on representative tissue cores of primary Gleason grade 3 and Gleason grade 5 tumor tissue. Adjacent tissue sections stained for basal epithelium (KRT5, green), pan-epithelial (pan-CK, red) markers, and hematoxylin and eosin (H&E) are included for comparison. Loss of green KRT5 was used to confirm areas of cancer. Cores were magnified and tiled. ****P < 0.0001. Original magnification, 20.
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High miR-182 in Low-Risk Prostate Cancer
miR-182 Is Expressed Exclusively in Prostate Epithelium and Is Higher in Areas of Cancer as Determined by ISH
The spatial expression pattern of miR-182 within the prostate was examined by ISH. Specificity of the miR-182 ISH probe was evaluated using RWPE1 cells over-expressing miR-182 and control cells19 grown as spheroids and formalin-fixed, paraffin-embedded processed to mimic the tissue conditions. miR-182 intensity was objectively quantified using the Vectra imaging system with inForm software version 2.4.1 (PerkinElmer) and higher in RWPE1 miR-183 clustereexpressing lentivirus spheroids (Figure 1A). ISH in formalin-fixed, paraffin-embedded radical prostatectomy tissues revealed that miR-182 was localized to the epithelium and was absent from stroma compared with the positive ISH control, which was present in all cell types (Figure 1B). miR-182 levels appeared higher in areas of cancer compared with adjacent benign epithelium within the same core (Figure 1C). Epithelial specific and high expression of miR-182 in PCa is consistent with