PDF - 1412KB, Supplementary Figure S1. 50 nM 1,25D is not growth inhibitory in PrE cells. Supplementary Figure S2. Mean levels of miR-100 and miR-125b expression by individual qRT-PCR in DU145, PC3, and commercially available PrEC cells treated with 50 nM 1,25D for 24H. Supplementary Figure S3. miR-100 and miR-125b is negatively correlated with targets PLK1 and E2F3 respectively in PrE cells. Supplementary Figure S4. No change in basal levels of miR-100 and miR-125b and their targets in the normal/cancer prostate cell line pair RWPE-1 and -2. Supplementary Figure S5. MiR-125b decreases migration of PrE cells. Migration was measured 0-48H after scratch in PrE cells transfected with pre-miRs (negative, -100, -125b). Supplementary Figure S6 Basal VDR levels of PrE, LNCaP, RWPE-1, and RWPE-2 cells. Supplementary Figure S7. EZH2 expression in benign and PCa patient tissue. Supplementary Figure S8. Let-7a is decreased in PCa and is positively correlated with prostatic 1,25D in patient tissue. Table S1. TaqMan Assay IDs for PCR. Table S2. miRNAs altered by 1,25D in primary prostatic PrE cells. Table S3. 1,25D-regulated miRNAs changes in PrE cells. Table S4. miR-100 and miR-125b alter growth of PrE, LNCaP, and RWPE-2 cells. Table S5. miRNAs are downregulated in PCa. Table S6. Spearman correlation of miRNAs to vitamin D metabolites.
ARTICLE ABSTRACTMiR-100 and miR-125b are lost in many cancers and have potential function as tumor suppressors. Using both primary prostatic epithelial cultures and laser capture-microdissected prostate epithelium from 45 patients enrolled in a vitamin D3 randomized trial, we identified miR-100 and -125b as targets of 1,25-dihydroxyvitamin D3 (1,25D). In patients, miR-100 and -125b levels were significantly lower in tumor tissue than in benign prostate. Similarly, miR-100 and -125b were lower in primary prostate cancer cells than in cells derived from benign prostate. Prostatic concentrations of 1,25D positively correlated with these miRNA levels in both prostate cancer and benign epithelium, showing that patients with prostate cancer may still benefit from vitamin D3. In cell assays, upregulation of these miRNAs by 1,25D was vitamin D receptor dependent. Transfection of pre-miR-100 and pre-miR-125b in the presence or absence of 1,25D decreased invasiveness of cancer cell, RWPE-2. Pre-miR-100 and pre-miR-125b decreased proliferation in primary cells and cancer cells respectively. Pre-miR-125b transfection suppressed migration and clonal growth of prostate cancer cells, whereas knockdown of miR-125b in normal cells increased migration indicates a tumor suppressor function. 1,25D suppressed expression of previously bona fide mRNA targets of these miRNAs, E2F3 and Plk1, in a miRNA-dependent manner. Together, these findings show that vitamin D3 supplementation augments tumor suppressive miRNAs in patient prostate tissue, providing evidence that miRNAs could be key physiologic mediators of vitamin D3 activity in prevention and early treatment of prostate cancer. Cancer Prev Res; 6(5); 483–94. ©2013 AACR.