840 860
ASC-J9
1
[11–15] .The results revealed that silencing
Malat1
expression by
Malat1
-siRNA or degrading AR-v7 by ASC-J9
1
suppressed the growth of EnzR cells
( Fig. 5 Aand B). Of note,
Malat1
induction (to induce AR-v7) is insufficient to render
ASC-J9
1
resistance to PCa cells (Supplementary Fig. 5),
suggesting targeting AR-v7 protein by ASC-J9
1
play a key
role to suppress AR-v7-mediated EnzR-PCa progression.
We then established in vivo mouse models via subcutane-
ously xenografting EnzR (R1) cells (1 10
6
) into nude mice.
After 3 wk, 16 of 30 injected mice developed tumors. We
divided these 16 mice into three groups and injected Group
1 with
Malat1
-siRNA (
n
= 6; 10 mg/kg body weight every
2 wk), Group 2 with ASC-J9
1
(75 mg/kg body weight every
24 h;
n
= 6), and Group 3 with anti-GFP oligonucleotides
[(Fig._3)TD$FIG]
Fig. 3 –
Malat1
is indispensable for androgen receptor splicing variant 7 (AR-v7) production. (A) Left, the efficiency of knocking down
Malat1
. Right,
AR-v7 expression in enzalutamide-resistant (EnzR) C4-2 cells was reduced in response to small interfering
Malat1
of two different sequences. (B)
Knockdown of
Malat1
attenuates Enz-induced AR-v7 production in VCaP cells. Cells were transfected with si
Malat1
and si-control (siCtr) for 24 h,
maintained in 1 nM dihydrotestosterone (DHT) with/without 10
m
M Enz for 24 h, then collected for full length AR (AR-FL) and AR-v7 detection by anti-
AR N-20 antibody. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. (C)
Malat1
and
AR
transcripts bind to serine/
arginine rich splicing factor 1 (SF2). RNA immunoprecipitation was performed using anti-SF2 antibody, the RNA in immunoprecipitate was extracted
with Trizol, followed by reverse transcription and detection with quantitative polymerase chain reaction for
Malat1
and
AR
transcript,
immunoglobulin-G (IgG) was used as a negative control. (D) EnzR cells have a higher SF2 activity. SF2 was immunoprecipitated from parental (P) and
EnzR C4-2 cells followed by immunoblotting with anti-SF2 antibody and anti-phospho-serine antibody. (E) Knockdown of SF2 blocks Enz-induced AR-
v7 production in VCaP cells. VCaP cells were infected with short hairpin (sh)RNA-SF2 lentivirus for 24 h then maintained in 1 nM DHT with/without
10
m
M Enz for 24 h followed by AR-FL and AR-v7 protein detection. GAPDH served as loading control.
[5_TD$DIFF]
(F
[6_TD$DIFF]
) Knocking down SF2 can decrease AR-v7 level
in EnzR cells. GAPDH served as a loading control.
Ser = serine.
E U R O P E A N U R O L O G Y 7 2 ( 2 0 1 7 ) 8 3 5 – 8 4 4
840