Pregm media

Primary prostate epithelial cells were isolated from deidentified radical prostatectomy tissue specimens as previously described under UIC Office for the Protection of Research Subjects–approved Institutional Review Board 2011-1138.¹⁴ (link) 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 −ΔΔCt method, using β₂-microglobulin for normalization.Table 1

Primers for Quantitative RT-PCR Validation of Predicted miR-182 Gene Targets

Gene	Primers
CD164	FWD	5′-TTAGCTTTCTCCCGAACGCC-3′
	RVS	5′-GCAGCTGTTTCGACCTTCAC-3′
ELL2	FWD	5′-ATGTGAAGCTCACCGAGACG-3′
	RVS	5′-TTTGACAAGCCCGTGGAGTC-3′
PRKAR1A	FWD	5′-ACACCCAGAGAGGGACAGAGAA-3′
	RVS	5′-GAGCTCACATTCTCGAAGGCT-3′
RNF152	FWD	5′-AGACTCGGTGACAGATACAGAAAT-3′
	RVS	5′-TGCAGGTAATGGCAAGCTCA-3′
NR3C1	FWD	5′-GTTGTTTATCTCGGCTGCGG-3′
	RVS	5′-TCAGTGAATATCAACTCTGGCA-3′
B2M	FWD	5′-CCTGAATTGCTATGTGTCTGGG-3′
	RVS	5′-TGATGCTGCTTACATGTCTCGA-3′

FWD, forward; RVS, reverse.

Prostatectomy tissues were provided by Dr Theodorus Van der Kwast at the University of Toronto. The patients were participants in a double‐blind randomized clinical trial of vitamin D₃ supplementation (Clinical trial CT00741364 http://www.clinical‐trials.gov) and deidentified.20 All patients in for sequencing were Caucasian and had a Gleason score of 3 + 3 or 3 + 4. For a 3 to 8 week period before surgery, patients in this study received 400 or 40 000 IU of liquid oral vitamin D3 daily. Prostatectomy tissue from the peripheral and transitional zones was freshly frozen and stored at −80°C. Serum 25D (cholecalciferol) and prostatic 1,25D (calcitriol) were measured by liquid chromatography‐tandem mass spectrometry and enzyme immunoassay, respectively, as part of the original trial (Table 1).20 Primary prostate epithelial cells (PrE) were isolated from deidentified benign prostatectomy tissue as determined by a pathologist and grown in PrEGM media (Lonza, Basel, Switzerland) for one passage on collagen‐coated plates17; the University of Illinois at Chicago (UIC) Institutional Review Board approved protocol #2011‐1138.

Deidentified prostate tissue and deidentified normal testis tissue were obtained through the UIC Biorepository (UIC IRB 2011‐1138). PrE were grown in PrEGM media (Lonza), for one passage. Primary stromal cells (PrS) were isolated under the same IRB as PrE as described,47 grown in MCDB media (Sigma) for 1 to 3 passage. RWPE1, PC‐3, LNCaP, 22RV1, and LAPC4 were obtained through ATCC (Manassas, VA) and grown less than 20 passages. RWPE1 were grown in KSFM media (Gibco, Dublin, Ireland), PC‐3 in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), LNCaP, 22RV1, and LAPC4 in RPMI (Gibco) with 10% FBS. Complementary DNA (cDNAs) were made with High‐Capacity cDNA RT Kit (Invitrogen). Primers are in Table 2. Quantitative PCR (qPCR) used SYBR green (Roche, Basel, Switzerland) and QuantStudio 6 (Thermo Fisher Scientific). PCR settings: 95°C 10 minutes (×1), 95°C 15 seconds, 58°C 30 seconds, 72°C 30 seconds (×40). Relative quantity was calculated by the −ΔΔC_t method, using GAPDH or HPRT1 for normalization.