JBC Papers in Press. Published on June 24, 2016 as Manuscript M116.718536 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M116.718536
lncRNA SOCS2-AS1 and prostate cancer
Androgen-induced lncRNA SOCS2-AS1 Promotes Cell Growth and Inhibits Apoptosis in Prostate Cancer Cells Aya Misawa1, 4, Ken-ichi Takayama1, 2, 4, Tomohiko Urano1, Satoshi Inoue1, 2, 3 1
Department of Anti-Aging Medicine, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan 2
Functional Biogerontology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan
3
Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama, 350-1241, Japan 4
Both authors contributed equally to this work.
To whom correspondence should be addressed: Prof. Satoshi Inoue, Department of Anti-Aging Medicine, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan. Phone: 81-3-5800-8834; Fax: 81-3-5800-9126; E-mail:
[email protected]. Keywords: androgen, androgen receptor, apoptosis, long noncoding RNA (long ncRNA, lncRNA), prostate cancer
(SOCS2-AS1),
ABSTRACT
whose
expression
higher in castration-resistant
Long noncoding RNAs (lncRNA)
was
prostate
have been associated with the development
cancer model cells, long-term androgen
of cancer. However, the interplay between
deprived (LTAD) cells than in parental
lncRNAs and androgen receptor (AR)
androgen-dependent
signaling in prostate cancer is still unclear.
SOCS2-AS1 promoted castration-resistant
Here, we identified lncRNAs induced by
and androgen-dependent cell growth. We
androgen in AR-positive prostate cancer
found
cells, whose induction was abolished by
upregulated genes related to the apoptosis
AR
an
pathway, including tumor necrosis factor
By
super family 10 (TNFSF10), and sensitized
knockdown
anti-androgen,
as
well
bicalutamide.
as
LNCaP
SOCS2-AS1
cancer
cells
cells.
knockdown
combining these data, we identified an
prostate
androgen-regulated lncRNA, suppressor of
treatment.
cytokine signaling 2-antisense transcript 1
demonstrated that SOCS2-AS1 promotes
Moreover,
to
docetaxel we
also
1
Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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Running title: lncRNA SOCS2-AS1 and prostate cancer
androgen signaling by modulating the
mechanisms for AR activation to improve the
epigenetic control for AR target genes
treatment of CRPC.
including
TNFSF10.
These
Some lncRNAs studied in prostate
findings an
cancer, act in a highly prostate-specific
important role in the development of
manner (13-18). In prostate cancer, the first
castration-resistant prostate cancer by
prominent lncRNA, PCA3, was initially
repressing apoptosis.
described as a novel biomarker of disease
suggest
that
SOCS2-AS1
plays
(19) and subsequently defined as a promising
technologies have greatly enhanced our
urine test for this disease (20). Similarly, the
understanding of the human transcriptome,
lncRNA PCGEM1 has been implicated in
revealing that more than 90% of the human
prostate cancer as a regulator of apoptosis
genome is actively transcribed, although only
(21). PCAT-1 was characterized as a novel
a minority is translated into proteins (1,2).
prostate-specific lncRNA, regulator of cell
Most of the non-coding transcripts more than
proliferation and target of the Polycomb
200
ncRNAs
Repressive Complex 2 (PRC2) (15). HOTAIR
(lncRNAs). (3). Although the number of
binds to AR protein to block the interaction
known human lncRNAs is still evolving,
with E3 ubiquitin ligase MDM2, thereby
some of them have been functionally
preventing protein degradation and AR
characterized and experimentally validated.
activation (22).
nt
are
known
as
long
Androgen receptor (AR) and its
In the previous study, we have
downstream signaling have a crucial role in
analyzed global AR transcriptional network
the development and progression of both
by mapping genome-wide transcriptional stat
localized and advanced metastatic prostate
sites (TTSs) regulated by androgen and AR
cancer (4-6). High-risk localized prostate
binding sites (ARBSs). This integrative
cancer is treated with androgen deprivation
genomic
therapies
and
AR-regulated transcripts from intergenic or
radiotherapy (7-9). However, many prostate
AS regions of genes in prostate cancer cells
cancers inevitably escape from androgen
(23). In addition, we investigated the
dependence leading to castration-resistant
functional
prostate cancer (CRPC) (10). Past studies
androgen-responsive long non-coding RNAs,
have revealed that elevated AR expression
such as a lncRNA located at the AS region of
(4), activation of AR transcription (11) and
C-Terminal Binding Protein 1 (CTBP1) gene,
development of AR variants (12) were
CTBP1-AS, revealing an oncogenic role of
observed in the progression to CRPC,
androgen-regulated lncRNAs in prostate
suggesting the importance of identifying AR
cancer progression (24).
in
addition
to
surgery
study
roles
revealed
of
comprehensive
these
novel
In the present study, to explore
downstream signals and new molecular 2
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Recent advances in sequencing
5α-dihydrotestosterone (DHT). In addition,
roles in prostate cancer progression, we
LNCaP and VCaP cells were also treated
performed
sequencing
with DHT plus bicalutamide, or 10 nM short
analysis in androgen-treated prostate cancer
interfering RNA (siRNA) targeting AR
cell lines. Among the androgen-induced
(siAR). After 24 hours, total RNAs were
lncRNAs in LNCaP and VCaP cells, we
extracted, and then, RNA-seq analysis was
focused on suppressor of cytokine signaling
performed. Bioinformatic analysis identified
2-antisense transcript 1 (SOCS2-AS1), whose
lncRNAs that were upregulated more than
expression
and
1.5 fold by DHT treatment, and repressed to
are
less than 0.75 fold by bicalutamide and siAR
cells
treatment in both LNCaP and VCaP cell lines.
derived from LNCaP and VCaP cells
Nine transcripts were common in both cell
(GenBank accession number: NR_038263).
lines using the GENCODE annotation and
SOCS2-AS1 promoted cell growth and
two in the NONCODE annotation (Fig. 1A).
migration, and repressed several genes
We examined these 11 transcripts and found
related to the apoptosis pathway, including
that they correspond to lncRNAs transcribed
TNFSF10, suggesting that androgen-induced
from five different genes (Table 1).
SOCS2-AS1 would play an important role in
SOCS2-AS1 is an androgen-induced lncRNA
the progression of prostate cancer.
highly
high-throughput
was
higher
VCaP-LTAD
in
cells
castration-resistant
prostate
LTAD
which cancer
expressed
in
castration-resistant
prostate cancer cells — Next, we performed RESULTS
qRT-PCR to analyze the expression of five
Identification of androgen-induced lncRNAs
lncRNAs in both LNCaP, VCaP and their
by directional RNA-sequencing — In order to
LTAD cells. We validated their androgen
investigate hormone-regulated lncRNAs in
induction as observed in RNA-seq data (Fig.
prostate cancer, we performed directional
1B and C). Among these five lncRNAs, we
RNA-sequencing (RNA-seq) and identified
found that SOCS2-AS1 was highly expressed
lncRNAs induced by androgen in prostate
in LTAD and VCaP-LTAD compared to
cancer cell lines. For lncRNA analysis, we
parental cell lines by RNA-seq and qRT-PCR
used two databeses, GENCODE V19 (25)
analysis (Fig. 1A-C, Fig. 2A). Therefore, we
and
v4
chose this lncRNA for further study. Our
positive
ChIP-seq data (26, 27) revealed AR binding
prostate cancer cell lines, LNCaP and VCaP,
site (ARBS) in the promoter region (Chr12:
and their corresponding castration-resistant
93,963,629-93,965,465 (hg19), +1.5 kbp -
cell
-291
NONCODE
(http://www.noncode.org/).
lines,
LTAD
AR
(long-term
androgen
bp
from
TSS
of
NR_038263).
deprived) and VCaP-LTAD cell lines (24)
Interestingly,
were treated with vehicle (ethanol) or 10 nM
repressed after 48 hour of androgen treatment 3
while
its
expression
was
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more androgen-dependent lncRNAs that have
in LNCaP cells, it was further induced even
SOCS2-AS1 expression level in LTAD cells
after 72 hours of androgen stimulation in
(Fig. 3C). In addition, AR binding to the
LTAD cells (Fig. 2B). Its sense gene, SOCS2,
promoter was observed in the absence of
showed a similar pattern of induction (Fig. 2
DHT and increased by low concentration of
B and C). These data indicates a distinct
DHT (Fig. 3D). These results indicate one
pattern of induction in castration-resistant
possibility that enhanced AR sensitivity in
prostate
LTAD cells induce the overexpression of
cancer
cells
compared
to
SOCS2-AS1.
hormone-naïve prostate cancer.
SOCS2-AS1 induces prostate cancer cell
transcribed from the opposite strand of the
growth — In order to investigate the role of
protein coding SOCS2 gene. SOCS2 is one of
SOCS2-AS1 in prostate cancer cells, we
the eight members of the SOCS family,
designed
which are induced by cytokine stimulation
androgen-induced
through
levels in LNCaP and LTAD prostate cancer
the
Janus
kinase
(JAK/STAT)
two
siRNAs
to
SOCS2-AS1
reduce expression
to
cell lines, as well as a SOCS2-targerting
cytokine inhibition by reducing JAKs or
siRNA (Fig. 4A, B and C). We found that
STATs phosphorylation, inhibiting the same
both SOCS2-AS1 and SOCS2 knockdown
cascade initiating their production through a
decreased cell proliferation in LNCaP and
negative feedback mechanism (28-30). We
LTAD cells (Fig. 4D). In addition, we
identified ARBSs at a common promoter
transfected siSOCS2-AS1 or siSOCS2 to
region of both SOCS2-AS1 and SOCS2, and
LNCaP and LTAD cells, and counted cell
in the intronic region of SOCS2-AS1,
number over time after androgen treatment.
suggesting that their expressions are directly
In line with the results of MTS assays, cell
regulated by AR (Fig. 2A). Consistently, AR
proliferation of siSOCS2-AS1 or siSOCS2
knockdown by siRNA and pre-treatment with
transfected cells was repressed compared to
the anti-androgen bicalutamide repressed
control siRNA transfected cells, supporting
SOCS2-AS1 and SOCS2 mRNA induction by
that SOCS2-AS1 and SOCS2 increase cell
androgen in both LNCaP and VCaP cells (Fig.
proliferation rate (Fig. 4E).
3 A and B).
We have previously reported
In addition, we established LNCaP cells
the enhancement of AR expression and
overexpressing SOCS2-AS1 or SOCS2 stably
sensitivity in LTAD cells compared with
(Fig. 5A and B). SOCS2-AS1 is located on
parental cells (31). We analyzed whether
chromosome
high sensitivity of AR to low concentration
(hg19)
of DHT is involved in the induction of
overlapping the promoter region of SOCS2
SOCS2-AS1 in these cell lines. We observed
(Fig. 2A). Given this close proximity, we
that low concentration of DHT increased the
hypothesized that SOCS2-AS1 may regulate
signaling.
SOCSs
genes
contribute
4
12
reverse
(93,959,404-93,965,174 strand,
NR_038263)
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SOCS2-AS1 is an antisense lncRNA
SOCS2. However, SOCS2 mRNA and
potential of prostate cancer cells.
protein
with
SOCS2-AS1 regulates the expression of
SOCS2-AS1 overexpression (Fig. 5B) and the
apoptosis associated genes such as TNFSF10
negative
— We performed microarray analysis to
levels
were
regulation
expression
by
not of
altered
SOCS2
antisense
protein
transcript
investigate
was
trans-regulatory
effects
of
marginally detected but not evident (Fig. 5C).
SOCS2-AS1 in LNCaP cells by comparing
We performed cell proliferation
the gene expression profiles in cells treated
assays using normal culture medium and
with siSOCS2-AS1, siSOCS2 or negative
androgen-deprived medium. We found that
control siRNA (siNC) (Fig. 7A). We then
increase
found SOCS2-AS1 and SOCS2 have distinct
of
cell
proliferation
rate
of
downstreams,
of SOCS2 stable cells, not only in the normal
biological responses by both genes were
medium
induced by such different gene regulations.
(Fig.
5D),
but
also
in
suggesting
different
androgen-deprived medium (Fig. 5E). These
Genes
results suggest that SOCS2-AS1 plays a
siSOCS2-AS1 transfection in DHT treated
central
both
cells were analyzed using the DAVID
hormone-dependent and castration-resistant
bioinformatics platform (32,33). Go-term
prostate cancer cell growth.
analysis showed an enrichment of genes
SOCS2-AS1 induces prostate cancer cell
involved in apoptosis (P = 4.4E-09) for 594
migration — Next, we performed migration with siSOCS2-AS1 or negative control siRNA.
upregulated by siSOCS2-AS1 transfection compared with Control siRNA in LNCaP cells (Fig. 7B).
Both cell lines were cultivated for two days
Interestingly, Tumor necrosis factor super
in androgen-deprived medium, transfected
family 10 (TNFSF10), which is involved in
with siRNA for 24 hours, transferred into
the apoptosis signaling was the most
8-µm pore seize inserts. The number of cells
upregulated
migrated among SOCS2-AS1 knockdown
transfection. Moreover, we also found genes
cells was lower compared to control cells
related with cell proliferation or cell cycle are
(Fig. 6A and B). We also performed
major signaling of SOCS2-AS1 downstreams.
migration assay using LNCaP cells stably
We further performed microarray analysis
expressing SOCS2-AS1, SOCS2 and control
using siSOCS2-AS1 transfected LTAD cells
cells. The number of cells migrated among
(Fig. 7C). We identified genes regulated by
SOCS2-AS1 overexpressing cells was higher
SOCS2-AS1 in LTAD cells (We obtained 301
than SOCS2 overexpressing cells or control
upregulated genes and 413 downregulated by
cells (Fig. 6C and D). These data suggest that
SOCS2-AS1 knockdown in the presence of
SOCS2-AS1 may enhance the metastatic
DHT). Interestingly, 37% of upregulated and
role
in
promoting
differentially
the
expressed
with
genes
assay in LNCaP and LTAD cells transfected
5
gene
by
siSOCS2-AS1
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SOCS2-AS1 stable cells was higher than that
81% of downregulated genes were found to
upregulated TNFSF10 expression in the
be LTAD specific SOCS2-AS1 targets (Fig.
presence of DHT in VCaP cells (Fig. 8C).
7D). GO-analysis of these genes indicates
Interestingly, TNFSF10 expression was
that SOCS2-AS1 regulates more variety of
totally repressed in LTAD cells (Fig. 8D, E)
signals
and
such
as
cell
migration
and
it
was
induced with
SOCS2-AS1
knockdown (Fig. 8F), suggesting the role of
apoptosis, cell cycle and cell proliferation
SOCS2-AS1 in repressing TNFSF10 in CRPC.
(Fig. 7E). We also found LTAD specific
Interestingly, most of other apoptosis-related
target genes associated with apoptosis such
genes in LNCaP cells (Fig. 8A) are also
as apoptosis-inducing factor 2 (AIF2) or
repressed in LTAD compared with LNCaP
checkpoint kinase 2 (CHEK2), suggesting the
cells and upregulated by siSOCS2-AS1 in
role of other signaling for apoptosis in LTAD
LTAD cells (Fig. 8G), suggesting the role of
cells.
SOCS2-AS1
upregulation
in
the
anti-apoptotic ability in CRPC cells.
TNFSF10 belongs to a small subset of pro-apoptotic protein ligands in the Tumor
SOCS2-AS1 inhibits apoptosis — In order to
necrosis factor (TNF) superfamily. TNF
examine whether SOCS2-AS1 is involved in
superfamily consists of proteins involved in
apoptosis, we performed cell proliferation
proliferation, differentiation and apoptosis,
assay in LNCaP and LTAD cells treated with
which also include TNF and Fas Ligand
docetaxel or DMSO, previously transfected
(FasL) (34,35). We also found that TRAIL
with siSOCS2-AS1 or siSOCS2. LNCaP cells
receptor 1 (DR4 or TNFRSF10A) as well as
were treated with 1 nM docetaxel, and LTAD
the cell death surface receptor Fas were also
with 2 nM docetaxel for 24 hours. In addition,
upregulated by siSOCS2-AS1 (Fig. 8A),
we showed further repression of SOCS2-AS1
indicating that SOCS2-AS1 may repress the
levels after 7 days of siRNA transfection (Fig.
apoptotic pathways. We confirmed that
9A). Cell viability was suppressed in the
TNFSF10 expression in LNCaP cells stably
presence of docetaxel in SOCS2-AS1 or
overexpressing SOCS2-AS1 was lower than
SOCS2
in control cells (Fig. 8B). In contrast,
SOCS2-AS1 knockdown LTAD cells (Fig 9B).
TNFSF10 expression was higher in LNCaP
Next, we performed Tunnel Assay with
cells treated with siSOCS2-AS1 knockdown
LNCaP and LTAD cells transfected with
cells (Fig. 8B). We confirmed by qRT-PCR
siSOCS2-AS1, siSOCS2 or siNC. Green
that TRAIL receptor 1 and Fas were
fluorescence-stained cells, representing cells
upregulated, supporting that SOCS2-AS1 may
undergoing apoptosis were observed in both
repress the apoptotic pathway by modulating
siSOCS2-AS1
the expression of these genes (Fig. 8B). We
LNCaP cells and siSOCS2-AS1 transfected
also
LTAD cells, whereas apoptotic cells were not
observed
siSOCS2-AS1
treatment 6
knockdown
and
LNCaP
siSOCS2
cells
and
transfected
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transcriptional regulation in addition to
significantly observed in control cells (Fig.
(35%) genes were induced (> 1.4 fold). This
9C, D). We further examined the role of
repression of androgen signaling was not
SOCS2-AS1 in apoptosis using VCaP cells
evident in SOCS2 knockdown samples. By
treated with DMSO or 2 nM docetaxel and
qPCR
obtained similar results (Fig. 9E, F).
androgen-dependent induction of ACSL3 or
Conversely,
analysis,
TMPRSS2,
SOCS2-AS1-overexpressing
both
we are
observed representative
cells showed resistance to docetaxel-induced
androgen-regulated genes, were enhanced in
inhibition of cell proliferation compared with
LNCaP cells overexpressing SOCS2-AS1 (Fig.
control cells (Fig. 10A). In addition, Tunnel
11B). In addition, we also confirmed that
Assay
SOCS2-AS1
using
SOCS2-AS1
and
SOCS2
knockdown
inhibits
these
androgen-mediated gene inductions in both
docetaxel
LNCaP and VCaP cells (Fig. 11C, D).
for
48h
showed
that
cells
overexpressing SOCS2-AS1 and SOCS2 were
Next, we further investigated whether AR
more resistant to apoptosis (Fig. 10B and C).
function is regulated by SOCS2-AS1. Among
Furthermore,
genes
western
blotting
analysis
upregulated
by
SOCS2-AS1
showed that the expression of cleaved-form
knockdown, we found AR-binding genes
PARP
in
were significantly enriched compared with
SOCS2-AS1 stably expressing cells compared
background by analyzing ChIP-seq data (Fig.
to control cells, when treated with 1 nM
12A). Then we explored the regulatory
docetaxel for 48 hours (Fig. 10D). Thus,
mechanism of AR signaling by SOCS2-AS1.
these data suggest that SOCS2-AS1 as well as
We found 8 genes out of 14 representative
SOCS2
genes
was
significantly
inhibit
apoptosis
reduced
and
promote
related
with
apoptosis
including
prostate cancer cell survival.
TNFSF10 have ARBSs in the vicinity. In the
SOCS2-AS1
androgen-mediated
intron region of TNFSF10, we found a strong
gene expression — We further analyzed
AR binding region and recruitment of CtBP2,
whether SOCS2-AS1 regulates AR activity
a cofactor important in prostate cancer
because we found more than half of
progression (28), to the ARBS (Fig 12B). In
upregulated
in
addition, androgen-dependent repression of
LNCaP cells were repressed by androgen
TNFSF10 was reversed by AR knockdown,
treatment and TNFSF10 is also repressed by
indicating the regulation of TNFSF10 by AR
androgen (Fig. 7A). We selected 559
(Fig. 12C). However, by western blot
androgen-induced (> 1.5 fold) genes. Among
analysis,
them, 267 (48%) genes were repressed (< 0.8
phosphorylated
fold) by siSOCS2-AS1 in the presence of
modification for AR binding to genome (36),
DHT (Fig. 11A). In addition, among 521
were not affected by siSOCS2-AS1 (Fig.
androgen-repressed genes (< 0.7 fold), 186
12D). In this ARBS, we observed histone
regulates
genes
by
siSOCS2-AS1
7
protein AR
levels
of
(ser81),
AR
or
important
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overexpressing cells treated with 2 nM
deacetylation and recruitment of HDAC to
TNFSF10 is repressed by androgen, whose
this region was inhibited by SOCS2-AS1
expression may be modulated by the action
knockdown (Fig. 12E, F), suggesting that
of SOCS2-AS1 on AR. In order to evaluate the importance of
SOCS2-AS1 is involved in AR activity by
SOCS2-AS1 in the clinical setting, we
modulating the epigenetic function. SOCS2-AS1
in
investigated the expression of SOCS2-AS1 as
cofactor
recruitments
—
well as its potential target, TNFSF10, in
Therefore, we investigated how SOCS2-AS1
clinical samples (38). SOCS2-AS1 was
regulates AR epigenetic function by ChIP
upregulated in some metastatic prostate
assay in three ARBSs (TNFSF10, ACSL3 and
cancer compared to clinically localized
TMPRSS2). Interestingly, androgen-mediated
primary prostate cancer, while TNFSF10
recruitment of CtBP2 was repressed by
inversely
SOCS2-AS1
AR
expression, supporting the oncogenic role of
bindings were not influenced (Fig. 13A).
SOCS2-AS1 (Fig. 14A and B). Taken together,
SRC1 is associated with gene induction by
our study provides a link between an
enhancing histone acetylation (37). We
androgen-induced lncRNA, SOCS2-AS1, and
observed recruitment of SRC1 only to
the apoptosis pathway, which may be
ARBSs of androgen-induced genes and
mediated by TNFSF10 (Fig. 14C).
involvement
AR-mediated
repressed
knockdown,
by
although
siSOCS2-AS1
correlated
with
SOCS2-AS1
treatment.
Androgen dependent histone acetylation was
DISCUSSION Previous studies have reported that several
also inhibited by siSOCS2-AS1 (Fig. 13B), SOCS2-AS1-mediated
lncRNAs are certainly vital for the molecular
epigenetic control in AR signaling could
chain of events that leads to prostate cancer
affect both AR-mediated gene induction and
development and progression. Therefore,
repression. Moreover, we analyzed whether
RNA-based therapy could be potential
SOCS2-AS1 is involved in AR activity by
strategy for CRPC. In the present study, we
interacting with AR. By qRT-PCR analysis,
investigated lncRNAs induced by androgen
we showed that SOCS2-AS1 is enriched in
in two AR-positive prostate cancer cells
the nuleus of LNCaP cells (Fig. 13C). By RIP
using directional RNA-seq method. Among
assay using nuclear lysates, we observed that
these lncRNAs, we found the expression
AR associates with SOCS2-AS1 in the
level of SOCS2-AS1 was higher in CRPC
presence of androgen (Fig. 13D). These
model
results suggest that SOCS2-AS1 interacts with
androgen-dependent LNCaP cells. Socs2
AR in the nucleus and modulates AR activity
knockout mice as well as transgenic mice
by
for
have been reported to display gigantism (39,
epigenetic controls. As an AR target gene,
40), suggesting a dual role for SOCS2 in
suggesting
regulating
that
cofactor
recruitment
8
LTAD
cells
than
in
parental
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of
The
we
and the effect of knockdown is relatively
SOCS2-AS1
small for inducing apoptosis in LTAD or
overexpression promoted castration-resistant
VCaP Tunel assay (Fig. 9D, E). Although
and androgen-dependent cell growth and
SOCS2-AS1 and SOCS2 are induced by
migration. On the other hand, SOCS2-AS1
androgen and knockdown of both decreased
knockdown inhibited cell growth of CRPC
cell proliferation rate, they affect different
model cells, as well as parental prostate
genes and pathways as suggested in the
cancer cells. Thus, our results indicate that
present study (Fig. 7A). These results support
SOCS2-AS1 may be a useful target for
that both, SOCS2-AS1 and SOCS2 confers
treating CRPC.
specific biological response to cell growth
growth
regulation.
demonstrated
Several
In that
studies of
have
study
addressed
bidirectional
and
the
apoptosis
by
modulating
different
downstream signaling.
transcription
(41-46). The expression of two genes from a
SOCS2-AS1 works as a regulator at
bidirectional promoter suggests a shared
the transcriptional level to affect the gene
upstream transcriptional regulator-mediated
expression profiles in several pathways
control of both genes, and implies that their
linked
functions might contribute to the same
apoptosis.
cellular response independently. Here we
apoptosis in cancer cells leaving normal cells
identified SOCS2-AS1 and SOCS2 as a pair
intact (47, 48), was found as a candidate of
of transcripts whose expression may be
SOCS2-AS1 target. TNFSF10 expression was
regulated by the same promoter/enhancer, in
repressed in CRPC cells and repression of
the opposite direction. Although our western
SOCS2-AS1 sensitized prostate cancer cells
blot analysis of SOCS2 in LNCaP cells
to
transfected with siSOCS2-AS1 suggests the
findings suggest that SOCS2-AS1 plays a key
putative negative sense/antisense regulation,
role in the development of CRPC by
further
repressing
experiments
about
stability
of
to
cancer
progression
TNFSF10,
docetaxel-induced
such
which
mediates
apoptosis.
TNFSF10
and
as
These
apoptosis.
transcript, for instance, are necessary to show
Furthermore, siSOCS2-AS1 downregulated
the relationship. Importantly, SOCS2-AS1
several known oncogenic genes known such
induction by androgen was concordant with
as Forkhead box protein M1 (FOXM1)
its sense strand gene, SOCS2, and both
and its target gene, Centromere Protein F
transcripts
the
(CENPF) which are highly expressed in
siAR
prostate cancer (49). FOXM1 encodes a
were
anti-androgen
repressed
bicalutamide
by and
forkhead
treatment. In
contrast
overexpression
to did
SOCS2-AS1, not
transcription
factor
involved in the regulation of cell growth,
SOCS2
enhance
domain
cell
proliferation and migration (Fig. 5, 6, 10A) 9
DNA damage,
genomic
resistance
metastasis.
and
stability, In
drug
addition,
Downloaded from http://www.jbc.org/ by guest on November 14, 2016
regulation
this
CENPF
12C). TNFSF10 was the representative gene,
activates PI3K and MAPK signal pathways
with a marked ARBS in the intronic region.
(50, 51). Together, these data support a role
We
for
cancer
SOCS2-AS1 on the AR pathway by analyzing
progression and provides a link of this
the effects of SOCS2-AS1 knockdown on the
lncRNA with cancer-specific genes.
expression of androgen-regulated genes.
coexpression
of
SOCS2-AS1
FOXM1
in
and
prostate
further
determined
the
effects
of
Microarray data indicate that SOCS2-AS1
been controversial (52-55). In prostate cancer,
depletion inhibited the androgen-mediated
it was reported that SOCS2 protein levels are
gene regulations (Fig. 11A). We further
reduced in CRPC, and that SOCS2 inhibits
analyzed the role of SOCS2-AS1 in AR
the oncogenic events caused by growth
binding and epigenetic regulation by ChIP
hormone (GH) such as cell proliferation and
assay.
invasion (56). In contrast, another report
modulate the cofactor recruitments and
revealed that SOCS2 protein expression was
histone modifications in TNFSF10 ARBS.
increased in prostate tumor tissues compared
Interestingly,
to benign tissues, and that SOCS2 has a
SOCS2-AS1 associated with AR by RIP assay
growth-promoting role in vitro and in vivo
in the nuleus. Thus, the transcriptional
(57). Our present study is in line with the
regulations by SOCS2-AS1 may be caused by
latter report, supporting the oncogenic role of
modulating AR epigenetic action. These
SOCS2 in prostate cancer progression, in
results suggest that high AR signaling in
concert with SOCS2-AS1. SOCS2 knockdown
castration-resistant prostate cancer induce
also repressed or induced genes related to the
strong SOCS2-AS1 expression, which can
apoptosis (data not shown), suggesting that
activate high levels of anti-apoptosis signals,
both transcripts may cooperate to inhibit
cell growth and lead to metastasis by
apoptosis and promote prostate cancer cell
enhancing AR signaling. Further studies will
survival.
be necessary to address the mechanisms of
We
demonstrated
we
also
SOCS2-AS1
observed
that
SOCS2-AS1 for transcriptional regulation.
Moreover, our microarray data
Recent
suggest that SOCS2-AS1 confers oncogenic
studies
have
signaling by transcriptional regulation of
several
genes associated with apoptosis, cell cycle,
whose
proliferation and migration. We found that
propensity of a tumor to metastasize. In this
genes related to apoptosis and upregulated by
study,
siSOCS2-AS1 transfection in DHT treated
potential of prostate cancer cells. A possible
LNCaP cells have ARBSs in the vicinity by
mechanism for such migration and cell
ChIP-seq analysis, suggesting that AR may
growth promoting ability of SOCS2-AS1 may
modulate the expression of these genes (Fig.
be the modulation of AR signaling by 10
metastases-associated
identified
overexpression SOCS2-AS1
can
lncRNAs, signify
enhanced
the
migration
Downloaded from http://www.jbc.org/ by guest on November 14, 2016
The role of SOCS2 in cancer has
SOCS2-AS1. As like several lncRNAs such as
androgen-dependent LNCaP cells, promotes
HOTAIR (22) and PCGEM1 (58), Our ChIP
castration-resistant and androgen-dependent
analysis indicated that SOCS2-AS1 may be
cell growth and cell migration. SOCS2-AS1
involved in the modification of AR for
modulated genes related to the apoptosis
recruiting
without
pathway, including TNFSF10, suggesting that
changing AR protein level or binding to
it may play a key role in the development of
genome. However, TNFSF10 is upregulated
CRPC by repressing apoptosis.
epigenetic
modifiers
even in the absence of DHT by SOCS2-AS1 EXPERIMENTAL PROCEDURES
possible
for
Cell culture and Reagents — LNCaP cells
SOCS2-AS1 such as the recruitment of other
were grown in RPMI medium supplemented
transcriptional factors to the chromatin site,
with 10% fetal bovine serum (FBS). VCaP
like CTBP1-AS (24), or the coactivation of
cells were grown in DMEM medium
cancer-related genes such as c-myc, like
supplemented with 10% FBS. LTAD cells
PCGEM1
were grown in phenol red-free RPMI
mechanisms
(59).
of
The
functions
investigation
of
associated transcription factors or epigenetic
medium
factors will be required to clarify how
charcoal-dextran–stripped FBS. For androgen
SOCS2-AS1
and
deprivation, cells were cultured for 3 days in
modulates AR signaling in prostate cancer.
phenol red-free RPMI medium (Nacalai
To show more molecular mechanisms of
Tesque,
SOCS2-AS1,
interaction
charcoal-dextran –stripped FBS. All the cells
partners by mass spectrometry would be
were maintained at 37oC in 10% O2 and 5%
interesting. Additionally, by comparing the
CO2. LNCaP and VCaP cells were purchased
results
from American Type Culture Collection.
of
interferes
with
investigating
microarray,
we
AR
identified
supplemented
Kyoto,
Japan)
with
10%
with
LTAD-specific target genes of SOCS2-AS1. A
DHT
previous study (6) has demonstrated that
bicalutamide (Casodex) were purchased from
different AR binding sites in CRPC model
Sigma (St. Louis, MO, USA).
cells from hormone naïve cells. Therefore,
RNA sequencing (RNA-seq) — RNA library
changes of AR or other transcription factor
preparation was performed according to the
binding sites in CRPC cells may influence on
manufacturer’s
the action of SOCS2-AS1, which interacts
OP-Illumina-Directional_RNA_seq-single_e
with AR for transcriptional regulation.
nd-v1.0 (Illumina, San Diego, CA, USA).
In summary, we show that an
(5α-Dihydrotestosterone)
2.5%
protocol
and
for
the
Sequencing libraries was constructed starting
SOCS2-AS1,
from 2 μg of total RNA. Sample DNA for
which is highly expressed in CRPC model
cluster generation was prepared according to
LTAD
the
androgen-induced cells
lncRNA, than
in
parental 11
manufacturer’s
protocol
for
the
Downloaded from http://www.jbc.org/ by guest on November 14, 2016
knockdown (Fig. 8A), suggesting other
genes using RefSeq Genes to determine the
RIP (RNA immunoprecipitation) assay —
exons genomic locations of known transcripts
was generated using PrimeScript RT reagent
Confluent LNCaP cells in 15-cm dishes were harvested and nuclear fractions were lysed in RIP buffer as described before (24). Specific antibody was added to the supernatant, and the mixture incubated overnight with rotation at 4°C. Thirty microliters of protein G beads was added, followed by incubation at 4°C for 2 h with gentle rotation. The beads were washed 3 times with lysis buffer and resuspended in 1 ml of ISOGEN (Nippon Gene). Co-precipitated RNA was isolated and used for qRT-PCR analysis.
kit (Takara, Kyoto, Japan). Expression levels
siRNA transfection — siRNAs were designed
were quantified by qPCR using KAPA SYBR
using
FAST ABI Prism 2X qPCR Master Mix and
(http://sidirect2.rnai.jp/) and purchased from
ABI StepOne system (Life Technologies,
Sigma
Gaithersburg, MD, USA). Relative mRNA
Cells were transfected with siRNAs using
levels were determined by normalization to
Lipofectamine
glyceralde- hyde-3-phosphate dehydrogenase
reagent (Thermo Fisher Scientific, Rockford,
(GAPDH) mRNA level. Primer sequences
IL, USA) at a final concentration of 20 nM,
are listed in Table 2.
according to the manufacturer’s protocol.
The
RNA-seq readings were analyzed using whole transcriptome software tools from Illumina.
Matching
locations
were
subsequently used to generate counts for
or coverage files (wiggle format). Finally, genes expression was determined as the number of reads per kilobase of exon model per million mapped reads (RPKM). RNA-seq data
was
uploaded
to
GEO
database
(GSE82225). Integrative Genomics Viewer version
2.2
(IGV
browser,
https://www.broadinstitute.org /igv/) was used for visualization. qRT-PCR — Total RNA was isolated using RNeasy Kit (QIAGEN). First strand cDNA
ChIP
(Chromatin
immunoprecipitation)
siDirect Genosys
ver.
2.0
(http://www.genosys.jp/). RNAiMax
transfection
siRNA sequences are listed in Table 3.
assay — ChIP was performed as described
MTS assay — Cells were plated at 2 × 103
before (23, 24). The fold enrichment
cells per well in 96-well plates. For RNA
relative to the GAPDH locus was quantified by qPCR using SYBR Green PCR master mix and the ABI StepOne System (Life Technologies, Carlsbad,
interference
experiments,
cells
were
transfected with 20 nM siRNA 24 h after plating. Cell viability was assessed at different time points using CellTiter 96 12
Downloaded from http://www.jbc.org/ by guest on November 14, 2016
annotated features, exons, transcripts or
CA). We used Myoglobin locus as a negative control (NC). Antibodies for ChIP have been described before. The primer sequences for ChIP qPCR have been described before (23, 24) or listed in Table 2.
OP-HiSeq2000-sequencing-cBot-v3.0.
AQueous One Solution Cell Proliferation
Tokyo, Japan).
Assay kit (Promega, Madison, WI, USA).
Migration Assay — Cell migration assays
Plates were incubated at 37°C for 50 min and
were performed using a 24-well plate
optical density was measured at 490 nm
matrigel-coated
using
microplate-spectrophotometer
Biosciences, San Jose, CA, USA). Briefly, 5
(Benchmark Plus, BioRad, Richmond, CA,
× 104 cells were suspended in serum-free
USA). Relative cell survival (mean ± SD of
RPMI
triplicate determinations) was calculated by
suspended 8 µm pore inserts. RPMI medium
standardizing the non-treated cell survival to
supplemented with 10% FBS was placed at
100%.
the bottom of the wells. After 24 h of
a
Cell proliferation assay — Cells were plated
medium
invasion
and
chambers
transferred
(BD
into
culturing, cells migrated to the opposite side
4
of the membrane were fixed with methanol
The following day, cells were transfected
and stained with Giemsa. The number of five
with 20 nM siRNA and cell number was
random fields were counted using Olimpus
counted at different time points as described
CKX41 microscope (×10 objective) and the
(24).
average number of cells per field was
Western blotting — Whole cell lysates were
calculated.
prepared using NP40 lysis buffer. Protein
Microarray analysis — For expression
concentration was measured by BCA assay
microarrays, a GeneChip Human Exon 1.0
(Pierce, Rockford, IL, USA). Cell lysates
ST Array (Affymetrix, Santa Clara, CA) was
were fractionated on 8% or 12% SDS–PAGE
used
gels, and transferred onto Immobilon-P
protocol as described (24). Data analysis was
Transfer Membrane (Millipore, Billerica, MA,
performed using the Affymetrix Microarray
USA). Membranes were incubated with
Suite
o
according
software.
to
the
To
manufacturer’s
compare
arrays,
primary antibodies at 4 C overnight and with
normalization was performed on data from
secondary antibodies at room temperature for
all probe sets. GO term and Pathway analysis
1 h, using Blocking One buffer (Nacalai).
was
Antibody-antigen complexes were detected
Microarray data was uploaded to GEO
with ECL Plus detection system (GE
database (GSE82225).
Healthcare,
Plasmid construction — To overexpress
Piscataway,
NJ,
USA).
performed
DAVID
SOCS2-AS1,
rabbit antibody #2779S (Cell Signaling,
specific primers which are listed in Table 3.
Danvers, MA USA), PARP rabbit antibody
cDNA synthesized using total RNA extracted
46D11
Signaling),
from androgen treated LTAD was used as
cleaved-PARP rabbit antibody D214 #9541S
template. We chose the most abundant PCR
(Cell signaling) and β-Actin #A5316 (Sigma,
product which corresponded to the length of
(Cell
13
performed
(25,26).
Antibodies used in this study are SOCS2
#9532S
we
using
PCR
with
Downloaded from http://www.jbc.org/ by guest on November 14, 2016
at 5 × 10 cells per well in 24-well plates.
SOCS2-AS1 variant 2 (ENST00000499137.2)
Tunnel
and cloned it into pcDNA3 expression vector.
DeadEnd™ Fluorometric TUNEL System
SOCS2 expressing vector was constructed by
(Promega). Briefly, slides were fixed with
transferring its protein coding sequence from
4% paraformaldehyde, permeabilized with
pDNR-LIB-SOCS2 vector (cDNA clone
0.2% Triton-X, equilibrated with buffer and
MGC:13697
to
stained with the reaction mix. The reaction
were
was stopped with 2 x SSC and cells were
Flag-tagged
IMAGE:4277425) pcDNA3.
Constructs
assay
was
performed
stained
1
mg/mL
Analyzer (Life Technologies).
4′,6-diamidino-2-phenylindole
(DAPI)
Establishment of SOCS2 and SOCS2-AS1
(Nacalai Tesque). Slides were mounted onto
overexpressing cells — SOCS2-AS1, SOCS2
glass slides and positively stained cells were
expressing vectors and empty vector were
counted with Olympus FluoView (FV10i)
transfected into LNCaP cells using FuGENE
microscope (×60 objective). Images were
HD (Promega) according to manufacturer’s
obtained through FluoView software (ver
protocol. Transfected cells were treated with
2.0)
500 µg/mL G418 (geneticin disulphate
Statistical
solution, Life Technologies) for selection of
proliferation assay, we analyzed 4 wells. For
stably transfected cells. Surviving colonies
the growth assay using stable cell lines, we
were picked up and grown as stable
performed two-way analysis of variance
transfected cells.
(ANOVA) at each time point. For other cell
Tunnel Assay — For RNA interference
line experiments, statistical differences (P
experiments, 5 × 104 LNCaP, LTAD or VCaP
values) among groups were obtained using a
cells were reverse transfected with 20 nM
two-sided Student’s t-test. All experiments
siSOCS2-AS1 (#1, #2), siSOCS2 or siNC
were performed at least twice and similar
(negative
results were obtained. Chi-square test was
and
suspended
on
analysis
to
—
analyze
For
the
the
ratios
cell
poly-L-lysine coated slides placed in each
performed
of
well of a 24-well plate. For SOCS2-AS1 and
AR-binding. P values less than 0.05 were
SOCS2 stably expressing cells, 5 × 104 cells
considered to be statistically significant.
were suspended on poly-L-lysine coated
Statistical procedures were performed using
slides placed in each well of a 24-well plate.
GraphPad Prism 5 software (GraphPad
24 h after plating, cells were treated with 1 or
Software, San Diego, CA, USA) or MS
2 nM docetaxel and cultured for another 24 h.
Excel.
Acknowledgements: The authors thank RIKEN for sequencing our samples. We are grateful to N. Sasaki and T. Oishi for technical assistance (microarray and bioinformatics analyses). We also thank to Dr. K. Ikeda (Saitama Medical University) for technical advice in siRNA design. 14
Downloaded from http://www.jbc.org/ by guest on November 14, 2016
sequenced using ABI PRISM 310 Genetic
control)
with
using
This work was supported by grants of the Cell innovation Program and the P-DIRECT from Ministry of Education, Culture, Sports, Science and Technology, Japan (SI); grants from Japan Society for the Promotion of Science, Japan (KT, grant 15K15581; SI, grant 15K15353); a grant of the Program for Promotion of Fundamental Studies in Health Sciences from NIBIO, Japan (SI); Grants-in-Aid (S.I.) from the MHLW, Japan; by Grants from Takeda Science Foundation (S.I. and K.T.); Grants from Mochida Memorial Research Foundation (K.T), Japan, the Yasuda Memorial Foundation (K.T.) and Princess Takamatsu Cancer Research Fund (K.T.)
Conflicts of interests: The authors declare no competing financial interests Author contributions: KT designed the study and performed RNA-seq. AM and KT performed manuscript.
15
Downloaded from http://www.jbc.org/ by guest on November 14, 2016
experiments and analyzed the data. SI supervised the study. AM, KT, TU and SI wrote the
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24
FIGURE LEGENDS Figure 1. Analysis of androgen-induced lncRNAs in prostate cancer and identification of SOCS2-AS1 as a lncRNA highly expressed in castration-resistant prostate cancer. (A) Flow diagram for the identification of SOCS2-AS1 as an AR-targeted lncRNA upregulated in LTAD cells.
Venn
diagram
representing
overlap
of
lncRNAs
induced
by
androgen
(10
nM
5α-dihydrotestosterone, DHT) and repressed by 1mM bicalutamide and 10 nM siAR treatment for 24 h in LNCaP and VCaP cell lines. (B) qRT-PCR Analysis of androgen-induced lncRNAs by qRT-PCR in LNCaP cells. (C) qRT-PCR Analysis of androgen-induced lncRNAs by qRT-PCR in VCaP cells. Cells were treated with 100 nM DHT or ethanol (Et) for 24 h. Expression level of each lncRNA was determined by qRT-PCR. N = 3. Expression levels are
Figure 2. SOCS2-AS1 is induced by androgen in LNCAP cells and is highly expressed in castration-resistant prostate cancer model LTAD cells. (A) RNA-sequencing (RNA-seq) and ChIP-sequencing (ChIP-seq) analysis of SOCS2-AS1 and SOCS2 in LNCaP and VCaP cell lines treated with 10 nM DHT or ethanol (Et) for 24 h. (B) Time course analysis of SOCS2-AS1 and SOCS2 mRNA after DHT treatment in LNCaP and LTAD cell lines determined by qRT-PCR. (C) Western blotting analysis of SOCS2 after 10 nM DHT treatment at indicated time points. β-Actin was used as an internal control. IB: immunoblot. Values represent mean ±s.d.; **, P < 0.01. Figure 3. AR knockdown and bicalutamide treatment represses SOCS2-AS1 expression. (A) qRT-PCR analysis of SOCS2-AS1, SOCS2 mRNA and AR mRNA levels following 10 nM siAR treatment and subsequent androgen treatment (10 nM DHT for 18 h) in LNCaP and VCaP cell lines (N = 3). (B) SOCS2-AS1 and SOCS2 mRNA levels following 1μM bicalutamide or dimethyl sulfoxide (DMSO) and 10 nM DHT or Et treatment for 24 h in LNCaP and VCaP cell lines by qRT-PCR (N = 3). RNA expression levels are presented relative to the value of GAPDH as reference gene. (C) qRT-PCR analysis of SOCS2-AS1 mRNA level after low dose of androgen treatment (0, 0.1, 1 nM DHT for 18 h) in LNCaP and LTAD cell lines (N = 3). (D) ChIP analysis of AR recruitments to the ARBS in the SOCS2-AS1 promoter region by low level of androgen. LNCaP and LTAD cells were treated with Et or 0.1 nM DHT for 24 h. 10 nM DHT was used as a positive control. Fold enrichment over control region (GAPDH) was measured by qPCR. Values represent mean ±s.d.; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Figure 4. SOCS2-AS1 knockdown inhibits LNCaP and LTAD cell growth. (A) Knockdown 25
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presented relative to the value of GAPDH as reference gene. Values represent mean ±s.d.
efficiency of SOCS2-AS1 by siRNA. LNCaP and LTAD cells were transfected with siNC:control siRNA or siSOCS2-AS1 #1 and #2 for 48 h and then treated with 10 nM DHT or Et for 18 h. Expression level of SOCS2-AS1 was analyzed by qRT-PCR (N = 3). (B) Knockdown efficiency of SOCS2 by siRNA, analyzed by qRT-PCR (N = 3) in LNCaP and LTAD cells treated with 10 nM DHT for 18 h. (C) Knockdown efficiency of SOCS2 by siRNA, analyzed by western blotting in LNCaP and LTAD cells, treated with 10 nM DHT or ethanol (Et) for 18 h. IB: immunoblot. (D) MTS assay in LNCaP and LTAD cell lines transfected with 20 nM siNC, siSOCS2-AS1 or 20 nM siSOCS2 for 24 h, following androgen treatment. (E) Cell proliferation assay in LNCaP and LTAD cell lines transfected with 20 nM siNC, siSOCS2-AS1 or 20 nM siSOCS2 for 24 h, following androgen (10 nM DHT) treatment. Values represent mean ±s.d.; *, P < 0.05; **, P < 0.01.
stable cells analyzed by qRT-PCR (N = 3). (B) qRT-PCR (N = 3) and western blotting analysis of SOCS2 expression in stable cell lines. IB: immunoblot. (C) SOCS2 protein expression analyzed by western blotting in LNCaP cells transfected with 20 nM siSOCS2-AS1 or siNC for 24 h, following androgen (10 nM DHT) or ethanol treatment for 18 h. IB: immunoblot. (D, E) MTS assays of stable cells cultured with normal medium (D) or phenol-free RPMI supplemented with 10% charcoal stripped FBS (E). Values represent mean ±s.d.; *, P < 0.05; **, P < 0.01. Figure 6. SOCS2-AS1 overexpression enhances cell migration. (A) Migration assay of siSOCS2-AS1 (#1, #2) and control siRNA transfected cells in 10 nM DHT medium. (B) Quantification of migrated cells. The number of four random fields were counted using a microscope and the average number of cells per field was calculated. (C) Migration assay of SOCS2 (#1, #2), SOCS2-AS1 (#1, #2) and control (#1, #2) stable cells. (D) Quantification of migrated cells. The number of four random fields were counted using a microscope and the average number of cells per field was calculated. Data represent the average of four different views. Values represent mean ±s.d.; *, P < 0.05; **, P < 0.01. Figure 7. SOCS2-AS1 inhibits the apoptosis pathway. (A) Microarray analysis of LNCaP cells transfected with 20 nM siSOCS2-AS1 (#2) or siNC for 24 h. Cells were treated with Et or DHT for 18 h. Genes repressed or induced more than 2-fold by siSOCS2-AS1 transfection in the presence of androgen were selected as SOCS2-AS1 downstream signals. Expression values displayed in gradient of red or green are the ratio between log2-transformed gene expression values in siSOCS2-AS1, siSOCS2 and siNC transfected cells. (B) GO-term analysis of 26
Downloaded from http://www.jbc.org/ by guest on November 14, 2016
Figure 5. SOCS2-AS1 overexpression enhances cell growth. (A) SOCS2-AS1 expression in
upregulated or downregulated SOCS2-AS1 signals. Most significantly enriched functions were presented. (C) Microarray analysis of LTAD cells transfected with 20 nM siSOCS2-AS1 (#2) or siNC for 24 h. Cells were treated with Et or DHT for 18 h. Genes repressed less than 0.7 fold or induced more than 1.6-fold by siSOCS2-AS1 transfection in the presence of androgen were selected as SOCS2-AS1 downstream signals. (D) Venn diagram showing the overlap of SOCS2-AS1 target genes in LNCaP and LTAD cells. (E) GO-term analysis of upregulated or downregulated SOCS2-AS1 signals in LTAD cells. Most significantly enriched functions were presented.
Figure 8. SOCS2-AS1 modulates TNFSF10 expression. (A) Expression levels of Representative genes, TNFSF10, FAS, TRAILR1 are shown by arrows. (B) TNFSF10 mRNA expression in SOCS2-AS1 stable cells analyzed by qRT-PCR. TNFSF10, Fas and TRAILR1 mRNA expression in 20 nM siSOCS2-AS1 (siSOCS2-AS1#2) or siNC transfected cells for 24 h in LNCaP cells. (C) TNFSF10 mRNA expression in 20 nM siSOCS2-AS1 (siSOCS2-AS1#2) or siNC transfected cells for 24 h in VCaP cells. (D) TNFSF10 mRNA expression in LNCaP and LTAD cell lines analyzed by directional RNA-seq using Integrative Genomics Viewer (IGV browser). (E) TNFSF10 mRNA expression in LNCaP and LTAD cell lines analyzed by qRT-PCR. (F) TNFSF10 mRNA expression in siSOCS2-AS1 transfected LTAD cells. Values represent mean ±s.d.; *, P < 0.05; **, P < 0.01. (G) (Left) Expression levels of apoptosis related genes (Fig. 8A) in LTAD cells were compared with LNCaP cells using microarray data. (Right) We compared expression levels of these genes in LTAD cells treated with siSOCS2-AS1with those in siNC. Figure 9. SOCS2-AS1 knockdown sensitizes cells to docetaxel treatment and increases apoptosis. (A) Knockdown efficiency of SOCS2-AS1 by siRNA, analyzed by qRT-PCR (N = 3) in LNCaP and LTAD cells treated with docetaxel for 7 days. (B) MTS assay of LNCaP and LTAD cells transfected with 20 nM siSOCS2-AS1, siSOCS2 or siNC for 24 h, following 1 nM docetaxel or DMSO treatment for indicated time points. (C, D) Tunel assay in LNCaP and LTAD cells transfected with 20 nM siSOCS2-AS1, siSOCS2 or siNC for 24 h following 1 or 2 nM docetaxel treatment for 24 h. (C) Panels represent 4′,6-diamidino-2-phenylindole (DAPI) (upper panels) and Alexa Fluor 488 (lower panels) signals detected in LNCaP cells treated with docetaxel. (D) Graph represents quantification of apoptotic cells in LNCaP and LTAD cells. (E) Tunel assay in VCaP cells. VCaP cells transfected with 20 nM siSOCS2-AS1, siSOCS2 or siNC for 24 h following 2 nM docetaxel treatment for 24 h. (F) MTS assay of VCaP cells 27
Downloaded from http://www.jbc.org/ by guest on November 14, 2016
apoptosis-related genes included in SOCS2-AS1 regulated genes in LNCaP microarray data.
transfected with 20 nM siSOCS2-AS1, siSOCS2 or siNC for 24 h, following 2 nM docetaxel or DMSO treatment for indicated time points. Data represent the average of three different views. (N = 3). Values represent mean ±s.d.; *, P < 0.05; **, P < 0.01 (vs siNC). Figure 10. SOCS2-AS1 overexpressing cells are more resistant to docetaxel treatment and apoptosis. (A) MTS assay of LNCaP stable cells treated with 2 nM docetaxel or DMSO for indicated time points. (B) Tunel assay in LNCaP stable cells treated with 2 nM docetaxel for 48 h. Panels represent 4′,6-diamidino-2-phenylindole (DAPI) (upper panels) and Alexa Fluor 488 (lower panels) stained docetaxel treated stable cells. (C) Quantification of apoptotic cells of Tunel assay. Data represent the average of three different views. (N = 3). Values represent mean ±s.d.; *, P < 0.05; **, P < 0.01. (D) Docetaxel-induced apoptosis is inhibited in SOCS2-AS1 expressing SOCS2-AS1 or SOCS2 and control cells, treated with 1 nM docetaxel for 48 hours. PARP and cleaved-PARP antibodies were used. PARP proenzyme (116 kD) and cleaved subunit, c-PARP, (89 kD) are indicated on the left by arrows. β-Actin was used as a loading control. IB: immunoblot. Figure 11. SOCS2-AS1 promotes androgen-mediated gene regulation. (A) Global effect of SOCS2-AS1 knockdown on androgen-mediated gene induction and repression. Microarray data from LNCaP cells transfected with 20 nM siSOCS2-AS1 (#2) or siNC for 24 h was analyzed. Among DHT-induced genes (> 1.5 fold), those repressed (< 0.8 fold) with siSOCS2-AS1 (#2) or siSOCS2 transfection are showed as the gray fraction (upper). Similarly, among DHT-repressed genes (< 0.7 fold), those induced (> 1.4 fold) with siSOCS2-AS1 (#2) or siSOCS2 transfection are showed as the black fraction (lower). (B) LNCaP cells overexpressing SOCS2-AS1 or vector control cells were treated with Et or 10 nM DHT for 24 h. Expression levels of ACSL3 and TMPRSS2 were measured by qPCR. Fold inductions over vehicle (Et) control are shown. (C) LNCaP cells were treated with siNC or siSOCS2-AS1#1 for 24 h and then treated with Et or 10 nM DHT for 24 h. Expression levels of ACSL3 and TMPRSS2 were were measured by qPCR. (D) VCaP cells were treated with siNC, siSOCS2-AS1 or siSOCS2 for 48 h and then treated with Et or 10 nM DHT for 24 h. Expression levels of ACSL3 and TMPRSS2 were measured by qPCR. Values represent mean ±s.d.; *, P < 0.05; **, P < 0.01. Figure 12. Androgen-dependent epigenetic control at TNFSF10 ARBS is modified by SOCS2-AS1. (A) AR binding genes were significantly enriched in SOCS2-AS1 downstream signals identified by microarray analysis (Fig. 7A). AR binding genes were determined based on 28
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stably expressing cells. PARP cleavage was analyzed by western blotting in LNCaP cells stably
our AR ChIP-seq data in LNCaP cells (28). Among genes upregulated or downregulated by siSOCS2-AS1 treatment in LNCaP cells, the number of AR binding genes was counted. Chi-square test was performed to determine the P-value. (B) Identification of ARBSs in the vicinity of apoptosis-related genes. Results of AR ChIP-seq are shown in the genomic loci of representative genes, TNFSF10, TNFSF18 and TNFAIP3. In TNFSF10, the result of CtBP2 ChIP-seq data (31) is also shown. (C) qRT-PCR analysis of TNFSF10 mRNA following 10 nM siAR treatment and subsequent androgen treatment (10 nM DHT for 18 h) in LNCaP cell lines (N = 3). (D) LNCaP cells were transfected with siSOCS2-AS1 #1 or siNC for 48 h and then treated with Et or DHT for 24 h. Protein levels of phosphorylated AR (ser81) and total AR were analyzed by western blotting. IB: immunoblot. β-Actin was used as a loading control. (E) ChIP analysis of histone acetylation (AcH3) was performed in LNCaP cells. Cells were transfected over GAPDH exon locus were calculated by qPCR (N = 3). (F) ChIP analysis of histone deacetylase 1 (HDAC1) was also performed. NC: myoglobin locus (N = 3). Values represent mean ±s.d.; *, P < 0.05; **, P < 0.01. Figure 13. SOCS2-AS1 interacts with AR and controls the association of AR cofactors for histone modification. (A) ChIP analysis of AR, CtBP2 and SRC1 was performed in LNCaP cells. Cells were transfected with siNC or siSOCS2-AS1 #1 for 48 h and treated with Et or DHT for 18 h. Fold enrichments over GAPDH exon locus were calculated by qPCR. NC: myoglobin locus (N = 3). (B) ChIP analysis of histone H3 acetylation (AcH3) was performed in LNCaP cells. Cells were transfected with siNC or siSOCS2-AS1 #1 for 48 h and treated with Et or DHT for 18 h. Fold enrichments over Et control were calculated by qPCR (N = 3). (C) LNCaP cells were treated with Et or DHT for 18 h. Total RNA was extracted from nuclear and cytosolic fractions. Expression level of SOCS2-AS1 was determined by qRT-PCR (N = 3). (D) RIP analysis of AR was performed in LNCaP cells. Cells were treated with Et or DHT for 18 h. Nuclear lysates were obtained and then immunoprecipitation using AR antibody was performed (N = 3). Associated RNA was isolated and then RNA level of SOCS2-AS1 was determined by qRT-PCR. MB: Myoglobin. Values represent mean ±s.d.; *, P < 0.05; **, P < 0.01. Figure 14. SOCS2-AS1 and TNFSF10 expression in clinical samples. (A, B) SOCS2-AS1 and TNFSF10 expression in publicly available datasets (GEO accession # GSE3325). SOCS2-AS1 (A) and TNFSF10 (B) microarray data was comparatively plotted in clinically localized primary prostate cancer and metastatic prostate cancer. Box plots are shown with lower and upper bounds of the boxes representing the 25th and 75th quartiles, respectively. Values represent normalized RNA expression levels relative to the value of GAPDH as reference gene. *, P < 29
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with siNC or siSOCS2-AS1 #1 for 48 h and treated with Et or DHT for 18 h. Fold enrichments
0.05; **, P < 0.01. (C) Schematic model of SOCS2-AS1 androgen induction and cancer progression in prostate cancer.
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30
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Database
RP1-288H2.2
FAM13A-AS1-002
FAM13A-AS1-001
SOCS2-AS1-005
SOCS2-AS1-004
SOCS2-AS1-003
SOCS2-AS1-002
SOCS2-AS1-001
TCONS_00029024
CTB-167B5.2
CTB-167B5.2
RP1-288H2.2
FAM13A-AS1-002
FAM13A-AS1-001
SOCS2-AS1-005
SOCS2-AS1-004
SOCS2-AS1-003
SOCS2-AS1-002
SOCS2-AS1-001
Transcript Name
ENST00000585152.1
ENST00000547538.1
ENST00000500765.1
ENST00000511543.1
ENST00000549723.1
ENST00000551626.1
ENST00000547845.1
ENST00000500986.1
ENST00000499137.2
n377170
n341270
ENST00000585152.1
ENST00000547538.1
ENST00000500765.1
ENST00000511543.1
ENST00000549723.1
ENST00000551626.1
ENST00000547845.1
ENST00000500986.1
ENST00000499137.2
ID
chr7:87900206-87903065
chr7:87900207-87903065
chr12:52486269-52498081
chr4:89630940-89651254
chr4:89642866-89649837
chr12:93960515-93965066
chr12:93959360-93965174
chr12:93945209-93960955
chr12:93936240-93965544
chr12:93936239-93965544
chr21:42953358-42954625
chr7:87900206-87903065
chr7:87900207-87903065
chr12:52486269-52498081
chr4:89630940-89651254
chr4:89642866-89649837
chr12:93960515-93965066
chr12:93959360-93965174
chr12:93945209-93960955
chr12:93936240-93965544
chr12:93936239-93965544
Locus
+
-
-
-
+
+
-
-
-
-
-
+
-
-
-
+
+
-
-
-
-
-
Strand
-
-
-
-
FAM13A
FAM13A
SOCS2
SOCS2
SOCS2
SOCS2
SOCS2
-
-
-
-
FAM13A
FAM13A
SOCS2
SOCS2
SOCS2
SOCS2
SOCS2
Sense Strand Gene
1182
2859
2859
512
3210
2453
354
618
555
1655
3325
1182
2859
2859
512
3210
2453
354
618
555
1655
3325
0.1693
0.2248
0.2248
5.6580
0.0988
0.1149
0.3409
0.3796
0.1528
0.0760
0.5654
0.0000
0.0000
0.0000
0.6635
0.0887
0.1160
2.1786
1.2480
1.3896
0.6158
0.3369
Et
29.9912
30.7869
30.7869
65.0695
0.5959
0.3946
30.6916
17.6279
19.7518
7.9838
4.0296
0.8558
0.4622
0.4622
1.5663
0.7211
0.7876
9.2162
5.2792
5.8784
2.1971
1.1402
DHT
8.8549
10.6454
10.6454
22.7286
0.1918
0.0858
9.6933
5.5525
6.1828
2.4825
1.2633
0.2429
0.0223
0.0223
0.2907
0.4074
0.3676
2.3425
1.3418
1.4941
0.6424
0.3453
DHT Bic
9.6232
7.2389
7.2389
18.6027
0.2155
1.1556
7.2389
10.0214
5.7733
6.4103
2.3647
0.0000
0.0443
0.0443
0.0825
0.1217
0.0818
2.2895
3.9970
2.5494
0.9953
0.5240
DHT siAR
RPKM
Genecode
CTB-167B5.2
n341270
chr21:42953358-42954625
Noncode
Genecode
Noncode
CTB-167B5.2
n377170
Length
Table 1. List of androgen-induced lncRNAs. lncRNAs induced by more than 1.5-fold with 10 nM DHT compared to ethanol (Et) treatment, repressed to less than 0.75-fold by bicalutamide (Bic) and siRNA targeting androgen receptor (siAR) in LNCaP and VCaP cell lines are listed. RPKM: reads per kilobase per million mapped reads.
LNCaP
VCaP
TCONS_00029024
Table 2. List of siRNAs and PCR primers used for RNA knockdown, qRT-PCR, ChIP-qPCR analysis and SOCS2-AS1 expression vector construction. siRNAs siSOCS2-AS1
#1
siSOCS2-AS1
#2
siSOCS2
SOCS2 TNFSF10 GAPDH
Forward Reverse Forward Reverse Forward Reverse Forward Reverse
CCATACAGGTCAACTTTTCCACCAC CCAACCTCAGCTCTGCTCTCTT AACCGCTCTACACGTCAGCA TGGTAAAGGCAGTCCCCAGA AGACCTGCGTGCTGATCGTG CAAGCAATGCCACTTTTGGA GGTGGTCTCCTCTGACTTCAACA GTGGTCGTTGAGGGCAATC
PCR primers for cloning of SOCS2-AS1 Forward CTCAAGCTTAATTTGGGAACGAGATGCCCGGGGA Reverse CCCTCTAGATAGAAAGCTCCAGGTGTGTTTTATTTTA ChIP qPCR Primers SOCS2-AS1 ARBS TNFSF10 ARBS
Forward Reverse Forward Reverse
TGGGACCGACGTTTAACTCT GGGGTATCCTGAGGCTGTG TCCGTACAGCTTGTGTGGAG TTGGTGGTTAGGGATCAAGC
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PCR Primers SOCS2-AS1
UUAUCACUCAUCAUUUCAGAA CUGAAAUGAUGAGUGAUAAUG GACCUGUAUGGUCAUUAUCACUC GUGAUAAUGACCAUACAGGUCAA UAUAUUCUUCCAAGUAAUCUU GAUUACUUGGAAGAAUAUAAA
GENCODE
Cell lines: LNCaP, VCaP
A
Treatment: DHT 10 nM 24h DHT + Bicalutamide DHT + siAR
LNCaP
58
Directional RNA-seq analysis Mapping: GENCODE, NONCODE Identification of AR-targeted lncRNAs
VCaP
9
72
NONCODE VCaP
Androgen induction Fold > 1.5 Repressed by bicalutamide or siAR
LNCaP
2
11 candidates (5 genes) of AR-targeted lncRNAs 53 Upregulation in LTAD, VCaP-LTAD cells Compared with parental cells By RNA-seq and qRT-PCR
114
AR-regulated lncRNAs in LNCaP cells.
SOCS2-AS1
AR-regulated lncRNAs in VCaP cells.
B 40
RP1-288H2.2
30 20 10 DHT
Et
DHT
0 Et
DHT Relative expression
DHT
Et
0
Et
25
DHT
50
5 4 3 2 1 0 Et
DHT Relative expression
75
Et
DHT Relative expression
0 Et
Et
DHT
Et
0
6
DHT
10
12
n341270
n377170
FAM13A-AS1 18
Et
20
DHT Relative expression
SOCS2-AS1
LNCaP LTAD
LNCaP LTAD
LNCaP LTAD
LNCaP LTAD
LNCaP LTAD
SOCS2-AS1
FAM13A-AS1
n377170
n341270
RP1-288H2.2
qRT-PCR
2 0 DHT
Et
DHT
0
4
Et
10
6
DHT
20
8
Et
30
RP1-288H2.2 DHT Relative expression
n341270 40
Et
DHT Relative expression
Et
n377170
250 200 150 100 50 0
DHT
DHT
Et
Et
0
DHT
20
Et
40
FAM13A-AS1 50 40 30 20 10 0
Et
SOCS2-AS1 DHT Relative expression
60
DHT
Relative expression
C
30
Et
Relative expression
qRT-PCR
VCaP VCaPLTAD
VCaP VCaPLTAD
VCaP VCaPLTAD
VCaP VCaPLTAD
VCaP VCaPLTAD
SOCS2-AS1
FAM13A-AS1
n377170
n341270
RP1-288H2.2
Figure 1
A
ChIP-Sequencing and Directional RNA-Sequencing
Chr 12
93,940 kb
93,950 kb
35 kb
93,960 kb
SOCS2-AS1
ARBS K4Me3 For
DHT Rev Et DHT
VCaP
Et DHT
VCAP -LTAD
LTAD
LNCaP LNCaP ChIP-seq
SOCS2
DHT
Et
Et
For Rev For Rev For Rev For Rev For Rev For Rev For Rev
C Western blotting
B 3
SOCS2-AS1 ∗∗
2 ∗∗
1
∗∗ ∗∗
∗∗
∗∗
∗∗
0 0 6 18 24 48 72 0 6 18 24 48 72
Relative expression
Relative expression
qRT-PCR 12
∗∗
SOCS2 ∗∗
8 4
∗∗ ∗∗
∗∗
∗∗
LNCaP IB: SOCS2
0
24 48
22 KDa ∗∗
β-actin 42 KDa
0 0 6 18 24 48 72 0 6 18 24 48 72
hours after induction
hours after induction
hours after induction
hours after induction
LNCaP
LTAD
LNCaP
LTAD
LTAD IB: SOCS2 22 KDa
β-actin 42 KDa
Figure 2
DHT 10 nM (h)
DHT 10 nM (h) 0
24 48
qRT-PCR
120 80 40 0 siNC
AR
SOCS2
∗∗
Relative expression
Relative expression
LNCaP SOCS2-AS1
siAR
120
Relative expression
A
∗∗
80 40 0 siNC
siAR
120
∗∗
80 40 0 siNC siAR
VCaP
B
80 40 0 DHT siAR DHT siNC siCtr siAR
∗∗
∗∗
120
Relative expression
∗∗
120
AR
SOCS2 Relative expression
Relative expression
SOCS2-AS1
80 40 0 DHT DHT siNC siAR siNC siAR
120 80 40 0 DHT DHT siAR siNC siCtr siAR
C
qRT-PCR
qRT-PCR SOCS2-AS1
Relative expression
12
∗∗
8 4 DHT Bic
DHT DMSO
Et DMSO
0 Et
DHT Bic
DHT DMSO
Et DMSO
SOCS2
1.6
∗∗
∗∗∗
∗∗∗
1.2 ∗∗∗
0.8
∗∗
0.4
0 DHT (nM)
0
0.1
1
0
0.1
1
0
siControl LNCaP
VCaP
Figure 3
20
LTAD
AR ChIP
5
10 5
VCaP
DHT Bic
DHT DMSO
Et DMSO
0
Fold enrichment
∗∗
15
Et
DHT DMSO
Et DMSO
Et
25 20 15 10 5 0
D
SOCS2
∗∗
Relative expression
SOCS2-AS1
∗∗
4
∗∗
3 2 1
0 DHT (nM)
0
0.1
LTAD
0
0.1
siAR
LNC P
DHT Bic
VCaP Relative expression
∗
16 12 8 4 0
Relative expression
SOCS2-AS1
Et
Relative expression
LNCaP
0.1 LNCaP
10
#1
Relative expression
120 100 80 60 40 20 0
#2
SOCS2-AS1 ∗∗ ∗∗
120 100 80 60 40 20 0
#1
siNC siNC siSOCS2-AS1
LTAD
LNCaP
LTAD
SOCS2-AS1 ∗ ∗∗
qRT-PCR SOCS2 120 100 80 60 40 20 0
Relative expression
LNCaP
Relative expression
B
qRT-PCR
siNC siSOCS2
Western Blotting
C
#2
SOCS2
∗∗
Relative expression
A
siNC siNC siSOCS2-AS1
IB:
∗∗
120 100 80 60 40 20 0 siNC siSOCS2
LNCaP
LTAD
Et DHT DHT
Et DHT DHT
SOCS2 22 KDa β-actin
D
MTS Assay LNCaP
Day 0
1.6 1.4
LTAD
Day 3
∗∗
1.2 1 0.8 0.6
∗
∗∗
1.8
Day 5
∗∗
Day 0 Day 3 Day 5
Fold Change
Fold Change
∗∗
∗∗
42 KDa
1.2
0.6
0.4 0.2 0 siNC si-NC
si-SOCS2-AS1 siSOCS2-AS1
#1 siSOCS2 si-SOCS2
siNC si-NC
Cell Proliferation Assay LNCaP 80 Et
x 104 cells / well
E
0
#2
60
DHT ∗∗
∗∗
40 20 0
#1
#2
#1
∗∗
x 104 cells / well
#1
#2
siNC siSOCS2siSOCS2-AS1siNCsiSOCS2-AS1 siSOCS2-AS1 siNCsiSOCS2-AS1 siSOCS2 siNC siSOCS2siSOCS2 -AS1 -AS1 Day 1
Figure 4
Day 5
15
#2
si-SOCS2-AS1 siSOCS2-AS1
LTAD
si-SOCS2 siSOCS2
Et DHT
10 ∗∗
∗∗
#1
#2
∗∗
5 0
#1
#2
siNC siSOCS2siSOCS2-AS1siNCsiSOCS2-AS1 siSOCS2-AS1 siNCsiSOCS2-AS1 siSOCS2 siNC siSOCS2 siSOCS2 -AS1 -AS1 Day 1
Day 5
A
B qRT-PCR
qRT-PCR
SOCS2
D
Relative expression
Relative expression
SOCS2-AS1 2.5 2 1.5 1 0.5 0 #1
#2
#1
LNCaPLNCaP Vec -Vec
#2
#1
#2
IB:
2000
SOCS2
22 KDa
β-actin
42 KDa
1500 1000 500
C
0 #1
#2
#1
#2
#1
E
MTS Assay **
DHT
Et
#2
LNCaP- LNCaP- LNCaPVec SOCS2 LNCaP LNCaP SOCS2LNCaP AS1 -SOCS2 -SOCS2-Vec AS1
LNCaP- LNCaPLNCaP SOCS2LNCaP SOCS2 -SOCS2 -SOCS2AS1 AS1
10
#1 #2 #1 #2 #1 #2
2500
siSOCS2-AS1 IB:
siNC siNC #1
SOCS2
22 KDa
β-actin
42 KDa
MTS Assay
#2
(Androgen deprived) ∗∗
2
8
Day 2
6
Day 5 4
Day 7
Fold change
Fold change
1.5
Day 2 1
Day 5 Day 7
0.5
2
0
0
#1
#2
LNCaP LNCaP-Vec -Vec
Figure 5
#1
#2
LNCaPLNCaP SOCS2 -SOCS2
#1
#2
LNCaP LNCaP-SOCS2SOCS2-AS1 AS1
#1
#2
LNCaP-Vec LNCaP -Vec
#1
#2
LNCaPLNCaP SOCS2 -SOCS2
#1
#2
LNCaPLNCaP -SOCS2SOCS2-AS1 AS1
A
B
Migration Assay
∗∗
siSOCS2-AS1 #2
LTAD
LNCaP
#1
Migrated Cell Number
siNC
∗∗
80
∗∗
∗∗
60 40 20 0 #1 si-NC siNC
#2
#1
si-SOCS2si-NC siSOCS2- siNC AS1 AS1 LNCaP LNCaP
LNCaP Vector -Vec
#1
#2
LNCaP SOCS2 -SOCS2
LNCaP SOCS2-AS1 -SOCS2-AS1
∗∗
120 100 80 60 40 20 0 #1
#2
#1
#2
Vec SOCS2 LNCaP LNCaP LNCaP LNCaP -Vec -SOCS2 -SOCS2 -Vec
Figure 6
si-SOCS2siSOCS2AS1 AS1 LTAD LTAD
D
Migration Assay
Migrated Cell Number
C
#2
#1
#2
SOCS2-AS1 LNCaP -SOCS2AS1
A LNCaP Microarray
B
Downregulated genes siSOCS2 siNC -AS1#2 siSOCS2
GO:0006508~proteolysis
Upregulated genes siSOCS2 siNC -AS1#2 siSOCS2
GO:0006357~regulation of transcription GO:0007242~intracellular signaling cascade GO:0042981~regulation of apoptosis GO:0042127~regulation of cell proliferation
Et DHT Et DHT Et DHT
Et DHT Et DHT Et DHT repressed
GO:0006955~immune response
0
10
20
30
-Log10 P- value Go analysis of upregulated genes by siSOCS2-AS1 in LNCaP cells
Induced
GO:0065003~macromolecular complex assembly
C
GO:0051276~chromosome organization
LTAD Microarray Downregulated genes siSOCS2 siNC -AS1#2 siSOCS2 Et DHT Et DHT Et DHT
Upregulated genes siSOCS2 siNC -AS1#2 siSOCS2
GO:0006259~DNA metabolic process GO:0007049~cell cycle
Et DHT Et DHT Et DHT
GO:0000279~M phase
repressed
0
5
10
15
20
25
-Log10 P- value Go analysis of downregulated genes by siSOCS2-AS1 in LNCaP cells Induced
E
D Fold > 2.0
Fold > 1.6
LNCaP
LTAD
GO:0008629~induction of apoptosis GO:0048545~response to steroid hormone GO:0007243~protein kinase cascade
GO:0043933~macrom olecular complex subunit organization GO:0006259~DNA metabolic process
404
GO:0048870~cell motility
110
190
GO:0008285~negative regulation of cell proliferation
GO:0051276~chromos ome organization
GO:0016477~cell migration
Fold < 0.7 LTAD
Fold < 0.5 LNCaP
344
75
327
GO:0000279~M phase
GO:0006928~cell motion GO:0051090~regulation of transcription factor activity GO:0006955~immune response
GO:0007049~cell cycle
0
10 -Log10 P- value
Go analysis of downregulated genes by siSOCS2-AS1 in LTAD cells
Figure 7
20
0
5
-Log10 P- value Go analysis of upregulated genes by siSOCS2-AS1 in LTAD cells
10
15
20
B
Apoptosis related genes siSOCS2 siNC -AS1#2 siSOCS2
LNCaP qRT-PCR TNFSF10
TNFSF10 Relative expression
TNFSF10 /TRAIL
80 60 40 20 0 Et
DHT
Et
repressed
Fas
TNFRSF10A /TRAILR1
0.05 0 #1
#2
LNCaP-Vec LNCaP
-Vec
Et
DHT
siNC siNC
Relative expression
VCaP qRT-PCR
D TNFSF10
∗∗
1.5
Et
0.5
LNCaPLNCaP SOCS2-AS1 -SOCS2-
AS1
Et
DHT
siSOCS2siSOCS2AS1(3) AS1
Et
DHT
siNC siNC
DHT
siSOCS2siSOCS2AS1(3) AS1
21 kb 10 kb
LNCaP LTAD
DHT
Et
Directional RNA-Seq Chr 3
∗∗
1
#2
2.5 2 1.5 1 0.5 0
LNCaP Microarray
C
#1
TRAILR1 3 2.5 2 1.5 1 0.5 0
Relative expression
Induced
0.1
siSOCS2siSOCS2AS1(3) AS1
siNC siNC
FAS
DHT
∗
0.15
Relative expression
Et DHT Et DHT Et DHT
Relative expression
A
0 siNC
siSOCS2AS1 TNFSF10
E
LTAD
∗∗
Relative expression
Relative expression
TNFSF10
200
qRT-PCR
F
qRT-PCR
150 100 50 0
100
Figure 8
LTAD
Expression levels of apoptosis-related genes
∗∗
80
up down
60 ∗∗
40 20
LTAD vs LNCaP
0 #1
Et DHT Et DHT LNCaP
G
TNFSF10
si-NC siNC
#2
si-SOCS2-AS1 siSOCS2-
AS1
up down siSOCS2-AS1 vs siNC in LTAD cells
A 200 ∗∗
150
∗∗
C
∗∗
LNCaP TUNNEL Assay
∗∗
siSOCS2-AS1
siNC
#1 DAPI
100 50 0 siNC siNC
#1 #2 siNC siSOCS2siSOCS2- siNC AS1 AS1
LNCaP LNCaP
#1 #2 siSOCS2siSOCS2AS1 AS1
LTAD LTAD
7 days post-transfection
B MTS Assay
LNCaP
2.5
day1
day3
∗∗ Relative apoptotic cells
1.5 1
∗
∗∗
∗
0.5 0 #1 siNC
#2
#1
siSOCS2-AS1 siSOCS2-AS1
siSOCS2
siNC
∗∗
50
∗∗
40 30 20 10 0 #1
#2
siSOCS2-AS1 siSOCS2-AS1
DMSO
LNCaP TUNNEL Assay
D
day5
2 Fold change
siSOCS2
#2
Alexa Fluor 488
Relative expression
SOCS2-AS1
si-NC siNC
#2
sisiSOCS2 SOCS2 Docetaxel 1 nM 24h Docetaxel 1 nM 24 h
siSOCS2
Docetaxel
si-SOCS2-AS1 siSOCS2-AS1
LTAD day1
day3
LTAD TUNNEL Assay
day5 Relative apoptotic cells
Fold change
2.5 2 1.5 1
∗∗
∗ 0.5 0 #1
#2
siSOCS2-AS1 siSOCS2-AS1
siNC
#1 siSOCS2
DMSO
F
∗∗ ∗∗
40
Fold change
Relative apoptotic cells
VCaP TUNNEL Assay
30 20 10
2
∗∗
∗∗
20 15 10 5 0 #1
#2
siNC siSOCS2-AS1siSOCS2-AS1 siSOCS2 siNC siSOCS2-AS1
siSOCS2
Docetaxel
E 50
25
#2
siSOCS2-AS1 siSOCS2-AS1
siNC
30
Docetaxel 2 nM 24 h
VCaP MTS assay day1
1.5
day3
day5
∗
∗
1
∗
∗∗
∗∗
0.5
0 #1 siNC siNC
#2
siSOCS2-AS1 siSOCS2-AS1
Docetaxel 2 nM 24 h
0 siSOCS2siSOCS2 AS1
Docetaxel 2 nM 24h
Figure 9
#1 siNC
#2
siSOCS2-AS1 DMSO
#1 siSOCS2
siNC
#2
siSOCS2-AS1 siSOCS2-AS1 Docetaxel
siSOCS2
A
Docetaxel 2 nM
MTS Assay 5
Fold change
Fold change
4 3 2
1.5 day0
1
day3 day5
0.5
1 0
0 #1
#2
LNCaP -Vec
B
∗∗
2
DMSO
TUNNEL Assay
#1
#2
#1
#2
LNCaP -SOCS2AS1
LNCaP -SOCS2
#2
LNCaP -Vec
#2
LNCaP-SOCS2 #1 #2
**
D
LNCaP-Vec
#1
#2
LNCaP -SOCS2
#1 #2 LNCaP -SOCS2AS1
LNCaP-SOCS2-AS1 #1 #2
Alexa Fluor 488
DAPI
#1
#1
Relative apoptotic cells
C
**
80 60
IB:
#1 #2
40 PARP
20 c-PARP
0 #1
#2
#1
#2
#1
#2
LNCaPLNCaP- LNCaPLNCaP LNCaP LNCaP Vec SOCS2 -SOCS2SOCS2-Vec -SOCS2 AS1 AS1 Docetaxel 2 nM 48 h
Figure 10
116 KDa 116 KDa 89 KDa
β-actin
#1 #2
#1 #2
Androgen-induced genes siSOCS2 siSOCS2-AS1
LNCaP stable cells
B
Up down Others Androgen-repressed genes siSOCS2-AS1 siSOCS2 Up down
Fold induction by androgen
A
∗∗ ∗∗
4.5 3
ACSL3 1.5
TMPRSS2
0 #1
#2
#1
LNCaP -SOCS2AS1
LNCaP -Vec
Others
#2
C TMPRSS2
ACSL3
10 ∗∗
5 0 siNC
siSOCS2-AS1#1
Relative expression
Relative expression
LNCaP
15
∗
10
Et
5
DHT
0 siSOCS2-AS1#1
siNC
TMPRSS2 40 N.S
30
∗∗
∗∗
20 10 0 siNC
#1
#2
siSOCS2-AS1
Figure 11
siSOCS2
Relative expression
Relative expression
D VCaP ACSL3 3
∗∗
∗
∗
Et
2
DHT
1 0 siNC
#1
#2
siSOCS2-AS1
siSOCS2
A
B
ChIP-seq
Chr 3: TNFSF10
173720 kb
173710 kb
Signal ratio
% of genes
Rate of AR-binding genes ∗∗ 0.3 0.2 0.1
10 4 80
CtBP2 AR
0
0
TSS
Chr 6: TNFAIP3
Chr 1: TNFSF18 171280 kb
171290 kb
138230 kb AR
AR
TSS
TSS MACS peaks
Relative expression
C
138240 kb
MACS peaks
2 1.5
Et
D
qRT-PCR
LNCaP
TNFSF10 ∗
∗
DHT
IB: P-AR
1 0.5 AR 0 siNC
siAR
Et
β-actin
DHT
E
F
HDAC1 ChIP
AcH3 ChIP TNFSF10 ARBS Et
DHT
N.S
4 ∗∗ 2
0 siControl siNC
Figure 12
∗∗
2.5 Fold enrichment
Fold enrichment
6
siSOCS2-AS1#1
2
Et siNC DHT siSOCS2 Et DHT -AS1#1
1.5 N.S.
1 0.5 0 NC
TNFSF10
A Fold enrichment
AR ChIP 150 Vehicle
100
siNC
DHT 50
Vehicle siSOCS2-AS1#l
DHT 0 NC
ACSL3 TMPRSS2 TNFSF10
SRC1 ChIP Fold enrichment
∗∗
30 20
∗∗
∗∗
10 N.S.
N.S.
ACSL3
∗∗ ∗
2
N.S. N.S. N.S.
1 0
TMPRSS2 TNFSF10
B
NC
ACSL3
C qRT-PCR Relative expression
Fold enrichment
AcH3 ChIP
ACSL3 6 ∗∗
4 2 0
Fold enrichment
TMPRSS2 8 4 2 0 Vehicle siNC
DHT
Vehicle
DHT
siSOCS2-AS1
∗∗
3 1.5 0 Et
DHT
Cytoplasmic fraction 10 8 6
Et
DHT
Nuclear fraction
RIP assay MB SOCS2-AS1
∗∗
4 2 0 Et
DHT IgG-IP
Figure 13
TMPRSS2 TNFSF10
SOCS2-AS1
4.5
D
∗∗
6
N.S.
N.S.
0 NC
3
Relative expression
Fold enrichment
CtBP2 ChIP
Et
DHT AR-IP
A
B SOCS2-AS1 2.00
*
**
Relative RNA levels
Relative RNA levels
4.00
TNFSF10
3.00 2.00 1.00 0.00
1.00
0.00
Primary Metastatic
Primary
Metastatic
Cytoplasmic
C Nucleus
AR
DHT
DHT
lncRNA SOCS2-AS1
AR antisense sense
Epigenetic regulation TNFSF10 AR
Genes for cell proliferation and migration
Proliferation ↑↑ Migration ↑ Anti-apoptosis ↑↑
Figure 14
SOCS2
(protein)
Proliferation ↑ Anti-apoptosis ↑
Androgen-induced lncRNA SOCS2-AS1 Promotes Cell Growth and Inhibits Apoptosis in Prostate Cancer Cells Aya Misawa, Ken-ichi Takayama, Tomohiko Urano and Satoshi Inoue J. Biol. Chem. published online June 24, 2016
Access the most updated version of this article at doi: 10.1074/jbc.M116.718536 Alerts: • When this article is cited • When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts
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