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. Author manuscript; available in PMC: 2019 Nov 1.
Published in final edited form as: Clin Cancer Res. 2018 Dec 26;25(9):2755–2768. doi: 10.1158/1078-0432.CCR-18-3230

Figure 5. Epigenetic silencing of miR-338-5p and miR-421 via EZH2 in SPINK1 positive prostate cancer.

Figure 5

(A) OncoPrint depicting mRNA upregulation of EZH2 and SPINK1 in MSKCC cohort using cBioportal. In the lower panel shades of blue and red represents Z-score normalized expression for EZH2 and SPINK1.

(B) Box plot depicting SPINK1, miR-338-5p and miR-421 expression in EZH2 high (n=119) and EZH2 low (n=119) in PCa patients from TCGA-PRAD cohort

(C) Representative micrographs depicting PCa tissue microarray (TMA) cores (n=238) stained for SPINK1 by immunohistochemistry (IHC) and EZH2 by RNA in-situ hybridization (RNA-ISH). Top panel represents SPINK1 IHC in SPINK1 negative (–) and SPINK1 positive (+) patients. RNA-ISH intensity score for EZH2 expression was assigned on a scale of 0 to 4 according to visual criteria for the presence of transcript at 40X magnification. Bar plot show EZH2 expression in the SPINK1-negative and SPINK1+ patient specimens. P-value for Chi-square test is indicated.

(D) Genomic location for EZH2 binding sites on the miR-338 and FTX promoters and location of ChIP primers (top panel). ChIP-qPCR data showing EZH2 occupancy and H3K27me3 marks on the miR-338, FTX promoters, and MYT1 used as positive control in stable 22RV1-miR-338-5p, 22RV1-miR-421 and 22RV1-CTL cells.

(E) GSEA plots showing the enrichment of EZH2 interacting partners (Kamminga) in 22RV1-miR-338-5p and 22RV1-miR-421 cells.

(F) QPCR data showing expression of EZH2 and its interacting partners in the same cells as indicated.

Biologically independent samples (n=3) were used in panels (D) and (F); data represent mean ± SEM. *P≤ 0.05 and **P≤ 0.005 using two-tailed unpaired Student's t test.