a let-7a let-7b let-7c - cancer research · 2011-08-26 · let-7a let-7b let-7c let-7i mir-98...
TRANSCRIPT
let-7d let-7f
let-7g
let-7b let-7clet-7a
let-7i miR-98
let-7e
Supporting Figure 1
A
00.5
11.5
22.5
64
MCF-10A HBL-100 BT474 SKBr-3 MCF-7 T47D BT549
Rel
ativ
e ex
pres
sion
le
vels
of l
et-7
f/U6
HMECs
highly invasive
Flow invasive
00.5
11.5
2
68
MCF-10A HBL-100 BT474 SKBr-3 MCF-7 T47D BT549
Rel
ativ
e ex
pres
sion
le
vels
of l
et-7
d/U
6 highly invasive
HMECsElow invasive
Rel
at. E
xpr.
leve
ls o
f let
-7f/U
6
0.0
0.25
0.50
0.75
1.0P=0.860
P=0.803
let-7f
C
Normal(N=12)
Metastasis-free (N=9)
Metastasis-positive (N=8)
Rel
at. E
xpr.
leve
ls o
f let
-7d/
U6
0.0
0.25
0.50
0.75
1.0
Normal(N=12)
Metastasis-free (N=9)
Metastasis-positive (N=8)
P=0.899
P=0.910
let-7d
B
D
0123456789
MCF-10A HBL-100 BT474 SKBr-3 MCF-7 T47D BT549MDA-453
MDA-435S
MDA-231
MDA-468
HMECs
highly invasivelow invasive
Rel
ativ
e ex
pres
sion
of l
et-7
g/U
6
G
24h
60h
D Control let-7g mimic
MDA-MB-231
0h 0h
48h
96h
Control let-7g ASO
T47D
E
Supporting Figure 2
A
00.20.40.60.8
11.2
let-7g ASOControl
Rel
at. x
pres
sion
of
let-7
g/U
6
let-7g
U6
* P<0.01
*
B
let-7g
U6
00.20.40.60.81
1.2
Control let-7g mimic
Rel
ativ
e E
xpre
ssio
n of
le
t-7g/
U6
*
* P<0.01
96 h
0 h
48 h
Control let-7g ASO C
MCF-7
Fold
-Mig
ratio
n/In
vasi
on *P<0.001
0
1
2
3
4
Migration Invasion
Controllet-7g ASO
**
G
*P<0.001P=0.016
Fold
-Mig
ratio
n/In
vasi
on
00.20.40.60.81
1.21.4
Migration Invasion
Controllet-7g mimic
*
H
Control let-7g mimic
0h
18h
24h
F
MDA-MB-468
K
TIME (Weeks)
Tum
or V
olum
e(m
m3 )
0
400
800
1200
1600
0 0.5 1 1.5 2 2.5 3
Controllet-7g ASO
J
*P<0.01
**P<0.02
*
*
***
*
Supporting Figure 2
I
00.51
1.52
2.53
3.54
4.5
control lenti-pre-let-7g
control lenti-let-7g ASO
Rel
ativ
e ex
pres
sion
leve
ls o
f le
t-7g
/U6
MDA-MB-231 MCF-7-luc
*
*
*P<0.001
liver
met
. lu
ng m
et.
Control let-7g ASO
200X
200X
400X
100X
Control let-7g ASO
lung
met
bone
met
L
lung macromets
Control
let-7g
P (χ2 test)
7/7
0/8
0.015
lenti-control lenti-pre-let-7gM
N
O
lenti-control
lenti-pre-let-7g
0
5101520
25
30lenti-controllenti-pre-let-7g
Num
ber o
f mic
rom
ets
per s
ectio
n
*P<0.001
*
Supporting Figure 2
P
MCF-7-let-7g ASO
MCF-7 Control
GAPDH
GAB2
si-NC si-GAB2#2
GAB2
ß-ACTIN
E
00.2
0.40.60.81
1.21.4
Migration Invasion
si-NCsi-GAB2 #2
*P<0.001*
Fold
-mig
ratio
n/in
vasi
on
*F
0
1
2
3
4
5
FN1 MMP2 MMP9
Rel
ativ
e ex
pres
sion
Control si-FN1let-7g ASO let-7g ASO+ si-FN1
A
GAB2 3’UTRLCS2
hsa-let-7g
Human 5’-AUACGACAGAGCUUUUAACUGGUGCCUUCCC -3’Chimpanzee 5’-AUACGACAGAGCUUUUAACUGGUGCCUUCCC -3’Rhesus 5’-AUACAACAGAGCUUUUCACUGGUGCCUUCCC -3’Mouse 5’-AUGUGGCAAAGCUUUUGACUGAUGCUGCCGG-3’Rat 5’-AUGUGGCUAAGCUCUUGAUUGGUGCUGCUGG-3’Rabbit 5’-AUAUAGC----AGCUUGUAACUGGUACCUCCCU -3’
3’- UUGACAUG------UUUGAUGAUGGAGU -5’|: | | | |: | | :| :| | | | :
5’- GGGCAAUAUGUUGCC—UGUUCUGCUUCA-3’Rabbit5’- UGACAGUUGGUUGCCC-UGUUCUGCUUCA-3’Rat5’- UGACAGUUGGUCACCC-UGUUCUGCUUCA-3’Mouse5’- GGGCUGCACAUUGCC—UGUUCUGCUUCA-3’Rhesus5’- GGGCUGCACAUUGCC—UGUUCUGCUUCA-3’Chimpanzee 5’- GGGCUGCACAUUGCC—UGUUCUGCUUCG-3’Human
3’-UUGACAUGU----UUG---AU----GAUGGAGU-5’
FN1 3’UTR
hsa-let-7g|: : : :: : :|||| ||| ||| ||
B chr 11 position 78,128,766 LCS1 LCS 2
NC_000011.9
77,926,342
395CTACCTC
2465TGCCTT
LCS3
2824ACTACCTC
3976bp
TTAT ATTT TATTTMutation
LCS1/Mut1: 201-632LCS2/Mut2: 1994-2676
LCS3/Mut3: 2711-3244
LCS2+3: 1994-3434
Dchr 2 position 216,225,163
LCSNM_002026
216,300,895
220TGCTTC
1096bp
ATTGATMutation (221-226)
C
Supporting Figure 3
GE F G
hsa-let-7a: UGAGGUAGUAGGUUGUAUAGUUmfe: -19.2 kcal/mol
hsa-let-7b: UGAGGUAGUAGGUUGUGUGGUUmfe: -21.4 kcal/mol
hsa-let-7c: UGAGGUAGUAGGUUGUAUGGUUmfe: -19.2 kcal/mol
hsa-let-7d: AGAGGUAGUAGGUUGCAUAGUUmfe: -17.9 kcal/mol
hsa-let-7e: UGAGGUAGGAGGUUGUAUAGUUmfe: -17.8 kcal/mol
H
hsa-let-7f: UGAGGUAGUAGAUUGUAUAGUUmfe: -17.8 kcal/mol
hsa-let-7g: UGAGGUAGUAGUUUGUACAGUU mfe: -19.9 kcal/mol
hsa-let-7i: UGAGGUAGUAGUUUGUGCUGUU mfe: -24.5 kcal/mol
hsa-miR-98: UGAGGUAGUAAGUUGUAUUGUUmfe: -17.4 kcal/mol
hsa-let-7a: UGAGGUAGUAGGUUGUAUAGUUmfe: -25.3 kcal/mol
hsa-let-7b: UGAGGUAGUAGGUUGUGUGGUUmfe: -30.3 kcal/mol
hsa-let-7c: UGAGGUAGUAGGUUGUAUGGUUmfe: -24 kcal/mol
hsa-let-7d: AGAGGUAGUAGGUUGCAUAGUUmfe: -23.4 kcal/mol
hsa-let-7e: UGAGGUAGGAGGUUGUAUAGUUmfe: -25.8 kcal/mol
I
hsa-let-7f: UGAGGUAGUAGAUUGUAUAGUUmfe: -19.7 kcal/mol
hsa-let-7g: UGAGGUAGUAGUUUGUACAGUU mfe: -21.4 kcal/mol
hsa-let-7i: UGAGGUAGUAGUUUGUGCUGUU mfe: -24.1 kcal/mol
hsa-miR-98: UGAGGUAGUAAGUUGUAUUGUUmfe: -17.9 kcal/mol
012345678
0 0.5 1 1.5 2 2.5 3 3.5 4GAB2 expression
let-7
g ex
pres
sion r= 0.5193
P=0.036
BHMECs (n=2)tumorigenic/non-metastatic (n=5)metastatic (n=3)
C
let-7
dex
pres
sion
r= 0.3005P=0.187
01020304050607080
0 0.5 1 1.5 2 2.5 3 3.5 4GAB2 expression
HMECs (n=2)tumorigenic/non-metastatic (n=5)metastatic (n=3)
D
010203040506070
0 0.5 1 1.5 2 2.5 3 3.5 4
GAB2 expression
let-7
fexp
ress
ion r= 0.3032
P=0.200HMECs (n=2)tumorigenic/non-metastatic (n=5)metastatic (n=3)
FN1 expression
let-7
gex
pres
sion
r= 0.3704
P=0.042
012345678
0 2 4 6 8 10 12 14
EHMECs (n=2)tumorigenic/non-metastatic (n=5)metastatic (n=4)
FN1 expression
let-7
fexp
ress
ion
010203040506070
0 2 4 6 8 10 12 14
r= 0.2013
P=0.642
GHMECs (n=2)tumorigenic/non-metastatic (n=5)metastatic (n=3)
FN1 expression
let-7
d ex
pres
sion
01020304050607080
0 2 4 6 8 10 12 14
r= 0.2962
P=0.572
HMECs (n=2)tumorigenic/non-metastatic (n=5)metastatic (n=3)
F
A T47D
Rel
ativ
eLu
cife
rase
Act
ivity
00.20.40.60.81
1.21.4
psi-check2 psi-FN1 3'UTR psi-GAB2 3'UTR
Controllet-7dlet-7flet-7g **P<0.05
*P<0.02
*** **
*
Supporting Figure 4
Rel
ativ
e R
enill
a/Fi
refly
luci
fera
se a
ctiv
ity
00.2
0.40.60.8
11.2
1.41.6
psi-check2 psi-FN1 3'UTR psi-GAB2 3'UTR
Controllet-7alet-7blet-7clet-7dlet-7elet-7flet-7glet-7imiR-98
H
A
0
1
2
3
4
0h 6h 12h 24h 48h
E2 (Time)
Rel
ativ
e E
xpre
ssio
n of
le
t-7/U
6
let-7dlet-7f
0
0.4
0.8
1.2
1.6
2
0h 6h 12h 24h 48h
EGF (Time)
B
Rel
ativ
e E
xpre
ssio
n of
le
t-7/U
6
let-7dlet-7f
0123456789
0h 6h 12h 24h 48h
Rel
ativ
e E
xpre
ssio
n of
FN
1/G
AP
DH
E2(10 nM)
E
00.5
11.5
22.53
3.5
0h 6h 12h 24h 48h
Rel
ativ
e E
xpre
ssio
n of
FN
1/G
AP
DH
EGF(20 ng/ul)
F
E2 Control
let-7g mimic
GAB2 siRNA
- + -- ---
-++ +++
++ --
-
--- --
-+ + -- -+ + -- --+ +-- - ++ - -- - ++
0
1
2
3
4
5
Fold
-Mig
ratio
n/In
vasi
on
Migration Invasion
T47D
C ** *
**P<0.01
EGF Control
let-7g mimicGAB2 siRNA
- + -- --
-
-++ ++
+
+
+ --
-
--- --
-+ + -- -
+ + -- --+ +-- - ++ - -- - ++
0
D
1
2
3
4
Migration Invasion
T47D
Fold
-Mig
ratio
n/In
vasi
on
**
**
*P<0.01
0 6 12 (Hr)
β-ACTIN
FN1
E2 (10 nM) EGF (20 ug/ul)
FN1
β-ACTIN
0 6 12 (Hr)
VectorFN1 3'UTR FN1 3'UTR Mutant
Rel
ativ
e lu
cife
rase
act
ivity
NS
NSNS
NS
0
0.5
1
1.5
2
2.5
3
EtOH+N.C. E2+N.C. EtOH+let-7g E2+let-7g
G
*P<0.05
*
*
VectorFN1 3'UTR FN1 3'UTR Mutant
NS
NSNS NS
0
0.5
1
1.5
2
2.5
3
ETOH+N.C. EGF+N.C. ETOH+let-7g EGF+let-7g
H
Rel
ativ
e lu
cife
rase
act
ivity *P<0.05
*
*
Supporting Figure 5
I
012345678
E2 Control
let-7g mimicFN1 siRNA
- + -- ---
-++ +++
++ --
-
--- --
-+ + -- -+ + -- --+ +-- - ++ - -- - ++
Fold
-Mig
ratio
n/In
vasi
on Migration Invasion
**
***P<0.001
J
EGF
Controllet-7g mimic
FN1 siRNA
- + -- --
-
-++ ++
+
+
+ --
-
-
-- --
-+ + -- -
+ + -- --+ +-- - ++ - -- - ++
Fold
-Mig
ratio
n/In
vasi
on
MigrationInvasion
024
1017
34
**
*P=0.002
*P<0.001
Supporting Figure 5
0
0.2
0.4
0.6
0.8
1
1.2
E2 EGF
VehicleE2/EGF+vehicleE2/EGF+U0126
Rel
ativ
e E
xpre
ssio
n of
pr
i-let
-7g/
pre-β-
actin
P=0.841P=0.452
ALAS1 TLR9 TWF2 PPM1M WDR82 GLYCTK
pre-let-7g
100 kb0
Chr.3
ERα Binding Sites (according to Carroll et al. 2006)
Exons of indicated genes
c-Myc Binding Sites (according to Chang et al. 2008)
K
L
E2 EGF
GAB2
let-7g
p44/42 MAPK
MMP-2/-9
Invasion/Metastasis
Poor Prognosis
Myc
LIN28LIN28B
TRBP FN1
p44/42 MAPK
Supporting Figure 6