Supplemental Methods
Reagents
Lipofectamine 2000, Lipofectamine RNAiMax, siRNAs, microRNA mimetics and non-target
controls, TaqMan probes and stem loop primer sets were obtained from Life Technologies
(Grand Island, NY) and used according to the manufacturer’s protocols. Pan-histone deacetylase
(HDAC) inhibitor vorinostat and DNA methyltransferase (DNMT1) inhibitor, 5-azacitidine,
were from LC Laboratories (Woburn, MA). MDV3100 was kindly provided by Medivation (San
Francisco, CA). All drug stocks (unless noted) were prepared in 100% DMSO and stored frozen
in small aliquots at -20° to eliminate degradation of the drug from repeated freeze-thaw cycles.
R1881 was obtained from Sigma Aldrich (St Louis, MO), reconstituted in ethanol and aliquots
were prepared and stored at -20°C. Anti-AR, anti-SRC1, anti-SRC3, anti-c-Myc, anti-pAKT(Ser473), anti-AKT, anti-p-S6(Ser235/236), Anti-S6, and anti-cleaved PARP antibodies
were obtained from Cell Signaling (Danvers, MA). Anti-SRC2 and anti-Skp2 antibodies were
obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Anti β-Actin antibody was
obtained from Sigma Aldrich (St. Louis, MO).
Cell Culture
Human cells lines were obtained from American Type Culture Collection (ATCC, Manassas,
VA) via the Tissue and Cell Culture Core Laboratory at Baylor College of Medicine, where they
are regularly submitted for cell line authentication (by STR profiling) and mycoplasma testing,
and passaged for fewer than 6 months: LNCaP cells were cultured in RPMI1640 supplemented
with 10% FBS; LAPC4 cells were cultured in Iscove's Modified Dulbecco's Media (IMDM, Life
Technologies) plus 15% FBS, 1 nM R1881 and 2 mM of L-glutamine; VCaP cells were
maintained in DMEM high glucose (Life Technologies) with 10% FBS and 1 nM R1881. All
media were supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin. Cells were
maintained in a 5% CO2 incubator at 37°C. Exponentially growing cells were utilized for all
described studies.
Transfection of microRNA mimetics
All microRNA (miRNA) mimetics or non-target controls (miRvana, Applied Biosystems) were
transfected into LNCaP, LAPC4 and 22Rv1 cells at 30 nmol/L (final concentration) using
Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s instructions.
Cells were incubated with the mimetics for 48-96 hours as indicated in each experiment.
MTT assay
Cells were plated in 24-well plates in medium containing 10% FBS and allowed to adhere for 24
hours. Then, miRNA mimetics were added, and the cells were incubated for an additional 96
hours. Cell viability was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT, obtained from Sigma-Aldrich) and expressed as a percentage of the nontargeting microRNA (miR-NT)-transfected wells. All experiments were repeated at least twice,
with each experimental condition repeated at least in quadruplicate per experiment.
RNA isolation and RT-qPCR
Cells treated with vorinostat, 5-azacytidine, enzalutamide (MDV3100) or R1881 were harvested
into Trizol. RNA was isolated by the addition of chloroform to the Trizol lysate, vigorously
shaking the tube and centrifuging the tubes at 13,000 rpm (16,100 x g) for 3 minutes to separate
the layers. The top, aqueous layer was pipetted into a clean 1.5 mL tube and 1 mL of 100%
isopropanol was added to precipitate the RNA. Tubes were centrifuged at 13,000 rpm for 10
minutes at 4°C. The isopropanol was decanted and the RNA was washed with 70% molecular
grade ethanol for 5 minutes. The ethanol was decanted and the RNA was air-dried. RNAse-free
water was used to resuspend the RNA pellet. The RNA was quantified and utilized for reverse
transcription (RT) reactions. To quantify microRNA expression levels following drug treatments,
a stem loop primer and a TaqMan probe for each target microRNA was utilized (Life
Technologies). Quantitative PCR was conducted utilizing a TaqMan hydrolysis probe for each
target microRNA. Relative expression of each miR was normalized to the expression of RNU6B.
For reverse transcription of total cell RNA for gene expression studies, a High Capacity Reverse
Transcription kit (Life Technologies) and random hexamers were used according to the
manufacturer’s protocol. The resulting cDNA was utilized for quantitative RT-PCR analysis of
KLK3/PSA and TMPRSS2 gene expression and was performed with SYBR Green PCR master
mix and gene specific primers using a StepOne Plus Real-time PCR system. Primer sequences
are provided in Supplemental Table 1. Relative expression of each mRNA was normalized to the
expression of β-Actin.
Reverse Phase Protein Array (RPPA) footprint of the SiM-miRNAs in PC cells
LNCaP PC cells were transfected with miRNA mimetics for 48 hrs, and assayed by RPPA using
a panel of 157 antibodies (41 against phosphorylated epitopes, 4 against cleaved epitopes and
112 against total proteins) with the help of the Functional Proteomics/RPPA Core Facility (The
University of Texas M.D. Anderson Cancer Center, Houston, Texas). Protein expression was
quantified by MicroVigene and normalized using the R package SuperCurve1. We determined a
proteomics signature of the microRNAs using the t-test, p-value<0.05, and fold change
exceeding 1.10. Pathway analysis of corresponding genes for recurrently changed antibodies was
performed using the ConsensusPathDB 2 software package. Hierarchical clustering and statistical
analysis was performed using the R statistical analysis system.
SDS-PAGE and immunoblot analyses
LNCaP cells transfected with miR-NT or miR mimetics, were lysed with RIPA buffer. Lysates
were cleared by centrifugation at 16,100 x g for 10 minutes. Protein estimation was conducted
with a BCA kit (Thermo Scientific). Proteins were mixed with 6 X SDS-PAGE sample buffer
and boiled for 5 minutes at 100°C. Proteins were separated on polyacrylamide gels and
transferred to PVDF-FL (low fluorescence) membranes. Blots were blocked with LI-COR
blocking buffer at room temperature for 30 minutes. Primary antibodies were added and
incubated for 3 hours to overnight. Blots were washed with 1X TBST and incubated with IR680conjugated anti-mouse or IR-800-conjugated anti-rabbit secondary antibodies for 1 hour. Blots
were washed and scanned on a LI-COR Odyssey scanner (LI-COR, Lincoln NE) and processed
with Image Studio version 3.1 (LI-COR). All immunoblots were performed at least twice and
representative blots are shown.
Bioinformatic prediction for direct miRNA/mRNA binding
Potential binding of miRNAs to 3’UTR of genes is evaluated using multiple criteria such as
sequence similarity with miRNA seed, estimated free energy after binding, evolutionary
conservation of the binding sites, and others. To examine which of the proteomic effects
observed after transfection of SiM-miRNA mimetics could be explained via direct binding to the
corresponding mRNAs, we combined the prediction for the SiM-miRNAs using five leading
prediction engines, namely TargetScan3-6, PicTar
13
7, 8
, miRanda
9, 10
, mirDB 11, and DianaLab
12,
. We used the union of all five prediction algorithms, so a miRNA/mRNA interaction was
considered possible if predicted by even one of these five engines. This was done in order to
have high sensitivity for predicted interactions.
Integration of AGO HITS-CLIP and AGO PAR-CLIP data
Publicly available Argonaute HITS-CLIP and PAR-CLIP datasets were interrogated using the
Starbase compendium
14, 15
(http://starbase.sysu.edu.cn/). Enriched regions were intersected with
the genes of interest AR, SRC1 (NCOA1), SRC2 (NCOA2), SRC3 (NCOA3), and ROCK1 using
BEDTools16. MicroRNA seeds were matched to the peak regions using miRanda9 as described
17
.
Data sets utilized were GSE57855, GSE43909, GSE28865, GSE32109, GSE41437, GSE43573,
GSE41357, GSE42701, GSE43910, GSE43912, and GSE43911 17-31.
Comparison of SiM-miRNA expression with AR transcriptional activity in PC tissues
Gene expression datasets derived from LNCaP cells after silencing AR have been previously
reported
32
. Gene expression differences were inferred utilizing the t-test and imposing a fold
change exceeding 4/3x (p<0.05), using the R statistical system, resulting in an AR-dependent
gene signature. In the Taylor et al.
33
dataset, we utilized the expression of each gene from the
AR signature to calculate its respective z-score for each sample, relative to the normal prostate
gland specimens available in that cohort. We then computed the sum z-score of all the AR-
signature genes for each sample (the z-scores of downregulated genes were subtracted from the
z-scores of upregulated genes), as described previously
33
, resulting in a corresponding gene
signature activity score for each specimen. We next computed the Pearson Correlation
Coefficient (PCC, p<0.05) between these gene signature activity scores and the expression level
of each miRNA in the Taylor et al.
33
dataset, using the R statistical system as previously 34. A
gene expression dataset derived from LNCaP cells after re-expressing miR-135a has been
reported by Kroiss et al (GSE45620) 31. We evaluated the significant enrichment of AR-induced
genes (genes suppressed by siAR) in the miR-135a transcriptomic footprint using the Gene Set
Enrichment Analysis (GSEA) 35 method (q<0.25).
Analysis of epigenetic marks in prostate cells in vitro
ChIP Seq datasets for AR, GATA2, FOXA1, SRC1, SRC2, SRC3, CBP, p300 and Pol II have
been previously reported
32
. We also used ChIP-Seq data of H3K4me3 and H3K27me3 from
PrEC and LNCaP cells (GSE38685)
36
. For enhancer identification, we used H3K4me1,
H3K4me2, and H3K27ac ChIP-Seq data in LNCaP cells
37
. Sequenced reads were mapped to
the human genome UCSC build hg19/NCBI build 37 using BWA
38
. High-resolution genome-
wide maps were derived and visualized in the UCSC Genome Browser (http://genome.ucsc.edu/)
and using IGV software 39, 40. Read coverage at the genomic loci for miR-135a-5p, miR-221-5p,
miR-1, and miR-31 was computed using BEDTOOLS
16
. To determine enrichment of a signal
between PrEC and LNCaP cells at specific genomic loci, we first used a 50 kbp window both
upstream and downstream of the locus of interest to establish the background signal. Then, we
calculated the ratio [reads at regulation site, defined by enrichment of either H3k4me3 or
h3k27me3]/[reads within 50 kbp of the locus in both directions] by Fisher’s exact test using the
SciPy Python library 41.
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