br The expression of miR for both cancer and
The AUY 922 of miR-182 for both cancer and benign cores was available for 56 patients on the Outcome TMA, and the cancer/benign ratio of miR-182 was significantly asso-ciated with recurrence. Patients with a greater increase in miR-182 expression in cancer tissue over benign tissue were significantly less likely to experience biochemical recurrence (Figure 2E). The cancer/benign ratio was not significantly different by patient ethnicity, Gleason score, or stage, although it did trend toward decreasing with increasing pre-procedure PSA values (Supplemental Figure S3). The Mur-phy TMA patients with high cancer/benign ratios tended to not have invasive pathologic findings, although the TMA was not designed with power to assess this statistically (Supplemental Figure S4). The association between high miR-182 cancer/benign ratio in the Outcome TMA and lower
916 ajp.amjpathol.org - The American Journal of Pathology
High miR-182 in Low-Risk Prostate Cancer
Table 3 Multivariate Logistic Regression for Recurrence
Variable Odds ratio (95% CI) P value
3 þ 3 Reference
The miR-182 cancer/benign ratios were segmented into tertiles (low, medium, and high ratios). Gleason scores of 4 þ 3 and 4 þ 4 were com-bined into a single predictor because there was only one patient with a Gleason score of 4 þ 4. *P < 0.05.
risk of recurrence was also evident in a multivariate logistic regression model for recurrence using clinical data from the Outcome TMA (odds ratio Z 0.18; 95% CI, 0.03e0.89; P Z 0.04) (Table 3). Because of the case selection criteria used for the original matched case-control design of this array, clinical parameters that normally predict recurrence (such as Gleason score and pT stage) were not significantly associated with recurrence.
Gene Targets of miR-182 Expression Are Enriched in Aggressive PCa
To identify potential prostate-specific targets of miR-182, the correlation between miR-182 expression and microarray gene expression profiles from LCM benign prostate epithelium was analyzed in a second cohort of patients with PCa (Figure 3A). Benign tissue was used instead of PCa because PCa exhibits high heterogeneity in gene expression patterns40 and thus decreases the likelihood of identifying targets of miR-182. The microarray data were previously reported by our group (Gene Expression Omnibus; https://
www.ncbi.nlm.nih.gov/geo; accession number GSE91037).27 For this study, miR-182 expression was quantified by RT-qPCR of the RNA from the LCM-collected samples using three reference RNAs for normalization. For 21 patients with both miR-182 and profiling data, Spearman correlation coefficients were calculated between the relative expression of miR-182 (-DDCt method) and each gene (log2 Robust Multiarray Average) (Figure 3A, Supplemental Table S1).
This approach to identifying miR-182 targets was validated by our observation that genes negatively correlated with miR-182 in prostate epithelium were enriched for predicted targets
of miR-182 from TarBase, TargetScan, and miRDB by gene-set enrichment analysis (Figure 3B).32e35 The genes most strongly negatively correlated with miR-182 in benign prostate epithelium that are also predicted targets are listed in Table 4. Kyoto Encyclopedia of Genes and Genomes version 6.1 pathways enriched in miR-182 negatively correlated genes included protein degradation, protein export, oxidative phos-phorylation, neurologic disorders, and bacterial infection (Figure 3C).36 Genes negatively correlated with miR-182 were strongly enriched for a gene set published by Chandran et al37 that contains genes up-regulated in metastatic PCa compared with primary PCa, which further links lower miR-182 to aggressive disease (Figure 3D). Many of the predicted miR-182 target genes from Table 4, ELL2, CD164, RNF152, and PRKAR1A, were validated in two different patient-derived primary prostate basal epithelial cells overexpressing miR-182 (Supplemental Figure S5, Figure 3E). To investigate whether the correlations identified were also present in PCa, TCGA prostate adenocarcinoma whole prostate tumor sequencing data were used. Despite a stromal bias in TCGA prostate adenocarcinoma samples,40 a few of the predicted targets and nearly a third of the top negatively correlated genes with miR-182 in our data set were negatively correlated with miR-182 in TCGA (Supplemental Tables S2 and S3). PRKAR1A and NR3C1, which have been reported to have higher expression in aggressive PCa,41,42 were negatively correlated with miR-182 in TCGA and validated as miR-182 targets in two patient-derived prostate cell lines (Figure 3E). r>