Transcript Document

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.