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Sample Age type (yrs) PrEC ? BPH 74 BPH 72 BPH 63 BPH 59 BPH 62 PCa 55 PCa 66 PCa 57 PCa 64 PCa 68 CRPC 82 CRPC 80 CRPC 61 Operation Gleason type grade --TURP -TURP -TURP -TURP -TURP -LRP 3+4 LRP 3+4 LRP 3+4 LRP 3+4 ORP 3+4 chTURP 5+5 chTURP 5+4 chTURP 5+5 TNM stage ------PT2c PT2a PT3a PT3a PT2c N/A N/A N/A Supplementary Table 1 – Details of clinical prostate samples used for miRNA microarray analysis. TURP: Transurethral resection of prostate, LRP: laparoscopic resection of prostate, ORP: open resection of prostate, and chTURP: channel TURP. N/A: Not Applicable. BPH PCa CRPC Common to all High in SC 508 524 487 unchanged 17 5 40 High in CB 310 306 308 429 - 239 Supplementary Table 2 – Shared miRNA expression in BPH, PCa, and CRPC fractionated populations. Values indicate the total number of miRNAs in each category (Total miRNAs: 835). Note the generally higher number of miRNAs overexpressed in SCs. Supplementary Table 3 – Composite miRNA signatures of PCa relative to BPH (top panel) and CRPC relative to PCa (bottom panel) Supplementary Fig. 1 – Validation of the microarray expression pattern with qRT-PCR, performed on the same samples used for microarray profiling (n = 5 BPH and 5 PCa, each sample in triplicate). • Integration of miRNA-mRNA microarray data derived from patient-derived prostate samples revealed that 60 potential miR-548c-3p target genes are repressed in SCs even at RNA level (manuscript in preparation). • Literature analyses showed that higher expression of miR-548c-3p is associated with poor survival (1) • Analysis of our published work (2) revealed that stimulation/overexpression of several transcription factors (e.g. RXR, VDR, GR, TAZ, SRF, HSF1) can promote prostate stem cell differentiation. Bioinformatic analysis revealed that miR-548c can inhibit expression of all these transcription factors. We hypothesised that higher expression of miR-548c-3p should be necessary for SC maintenance in prostate epithelium Supplementary Fig. 2 – Rationale for selecting miR-548c-3p 15 * 10 5 TA C B 0 SC Relative miR-548c-3p expression Supplementary Fig. 3 Relative miR-548c-3p expression Expression of miR-548c-3p in prostate epithelial sub-populations. (Newly cultured patient samples distinct from those used in Fig 1 or Figure S1a. n = 5 BPH and 5 PCa, each sample in triplicate). Data are expressed as mean ± s.d. *P < 0.05 (Student's t-test). 8 *** 6 4 2 0 c p S C R - 5 48 c - 3 miR ** 3.6 3.1 ** 2.6 * 2.1 1.6 * 1.1 0.6 0.1 B FK N * * ID 2 PR O M 1 SO X2 C EA C A M 6 W N T5 A LC N 2 * 1 Relative qRTPCR expression Expression of miR-548c-3p CB cells 3 days after transfection with miR-548c-3p. (n = 2 BPH and 3 PCa, each sample in triplicate). Data are expressed as mean ± s.d. ***P < 0.001 (Student's t-test). Gene expression changes, as assessed by qRT-PCR, of differentiation-associated genes after miR-548c-3p transfection into CB cells for 3 days, relative to control transfection. (n = 2 BPH and 3 PCa, each sample in triplicate). Data are expressed as mean ± s.d . *P < 0.05, **P < 0.01 (Student's t-test). miR-548c-3p: Examples of potential target genes: HDAC3, SPOP, KLF4, MAZ, CDKN1B, and WNT5A GO: Molecular functions Chromatin binding Transcription factor activity RNA polymerase II regulatory region sequence-specific DNA binding GO: Biological processes Cell cycle Cell differentiation Cellular response to stress q value 9.454E-4 9.454E-4 4.543E-3 q value 2.552E-6 3.909E-6 3.909E-6 Top GO enrichment terms for potential miR-548c-3p targets Supplementary Fig. 4 – The potential miR-548c-3p targets were predicted using miRWalk algorithm and gene ontology analysis was performed on them. Note: Such in silico analysis has known shortcomings and further experimental proof is necessary to extend these conclusions. References: 1. Taylor B, Schultz N, Hieronymus H, et al. Integrative genomic profiling of human prostate cancer. Cancer Cell. 2010;18(1):11-33. 2. Rane JK, Droop AP, Pellacani D, et al. Conserved two-step regulatory mechanism of human epithelial differentiation. Stem cell reports. 2014;2(2):180-8.