supplemental table 1. oligonucleotide sequences used for mrna expression analysis by qpcr. primer...

Supplemental Table 1. Oligonucleotide sequences used for mRNA expression analysis by qPCR.

Primer name

Forward Primer 5’- 3’ Reverse Primer 5’-3’

KLK3 5’-GTGCTTGTGGCCTCTCG-3’ 5’-AGCAAGATCACGCTTTTGTTC-3’

TMPRSS2 5’-CGCTGGCCTACTCTGGAA-3’ 5’-CTGAGGAGTCGCACTCTATCC-3’

AR 5’- AAGGCTATGAATGTCAGCCCA-3’ 5’-CATTGAGGCTAGAGAGCAAGGC-3’

SRC1 5’-TGCAGCAGAATGTCTTCCAG-3’ 5’-AAAGTTGGCCTCACCTTGG-3’

SRC2 5’-ACCTGGTAAGAAGGTGTATTCAGAAG-3’ 5’-TGCCTCTTAGCATAGGACACAGA-3’

SRC3 5’-TCAACCCACTTCCTTCAGAGA-3’ 5’-ACATACTCATGCCGCCTACC-3’

ROCK1 5’-GAAACAGTGTTCCATGCTAGACG-3’ 5’-GCCGCTTATTTGATTCCTGCTCC-3’

β-Actin 5’-CACCATGTACCCTGGCATT-3’ 5’-GTACTTGCGCTCAGGAGGAG-3’

B

Supplemental Figure 1

A

miR

-205

miR

-1

miR

-133

a

miR

-133

b

miR

-143

-5p

miR

-145

-3p

miR

-204

miR

-143

-3p

miR

-31

miR

-24-

1-5p

miR

-221

-3p

miR

-135

a-5p

miR

-222

-3p

0

5

10

15

miR

-221

-5p

Primary CancerNormal Prostate

mic

roR

NA

exp

ress

ion

(log2

)

***

*

***

***

***

***

***

**

***

***

***

***


mic

roR

NA

exp

ress

ion

(log2

)

***

***

*** **

***

***

* ** *

miR

-205

miR

-1

miR

-133

a

miR

-133

b

miR

-143

-5p

miR

-145

-3p

miR

-221

-5p

miR

-204

miR

-143

-3p

miR

-31

miR

-24-

1-5p

miR

-221

-3p

miR

-135

a-5p

miR

-222

-3p

4

6

8

10

12

C

mic

roR

NA

exp

ress

ion

(log2

)


***

***

*** * ** *

* ** *

***

*

miR

-205

miR

-1

miR

-133

a

miR

-133

b

miR

-143

-5p

miR

-145

-3p

miR

-221

-5p

miR

-204

miR

-143

-3p

miR

-31

miR

-24-

1-5p

miR

-221

-3p

miR

-135

a-5p

miR

-222

-3p

−10

−5

0

5

D E


F

* p< 0.05 by one way ANOVA with Dunnsmultiple comparison test for each column

G

Supplemental Figure 1lo

gex

pre

ssio

no

fm

icro

RN

A

miR

-20

5

miR

-1

miR

-133

a

miR

-133

b

miR

-143

-5p

miR

-145

-3p

miR

-221

-5p

miR

-20

4

miR

-143

-3p

miR

-31

mIR

-24

-1-5

p

miR

-221

-3p

miR

-135

-a-5

p

miR

-222

-3p0

5

10

15Low Gleason Intermediate Gleason High Gleason

*

*

0 20 40 60 80 1001200.00.20.40.60.81.0

Top 75%Bottom 25%

p= 0.1211

BC

R-fr

ee s

urvi

val

miR-143-3p

Time (months)0 20 40 60 80 100120

0.0

0.20.40.60.81.0

Top 75%Bottom 25%

p= 0.0982

BC

R-fr

ee s

urvi

val

miR-204

Time (months)

0 20 40 60 80 100 1200.00.20.40.60.81.0

Top 75%Bottom 25%

p= 0.5056

BC

R-fr

ee s

urvi

val

miR-222-3p

Time (months)


0 20 40 60 801001200.00.20.40.60.81.0

Top 75%Bottom 25%

p= 0.4976

BC

R-fr

ee s

urvi

val

miR-24-1-5p

Time (months)

Top 75%Bottom 25%

p= 0.6361

BC

R-fr

ee s

urvi

val

miR-143-5p

Time (months)0 20 40 60 80100120

0.00.20.40.60.81.0


CPDB Pathways

p21

RTK

PI3

KIR

S1

PIP 2

PIP 3

PD

K1

mTO

RC

2A

KT

T308

S47

3

TSC

1/TS

C2

mTO

RC

1 p70S

6K4E

BP1

FO

XO

3A

p21

p27

BIM

S6

eiF4

E

PRA

S40

p53

CD

C2

Rb

CD

K4

A

R

SRC

3

PTE

N

Cel

l Cyc

le/G

1-S

Tran

sitio

n

INPP

4B

AR

SRC

3

BIM

CD

C2

CD

K4

p27

INPP

4B

p4EB

P1 S

65p4EB

P1 T

37/4

6

p53

pRB

S80

7/81

1p-

S6 S

240/

244

p-S6

S23

5/23

6p-p7

0S6K

PTEN

p-PR

AS4

0

Supplemental Figure 4 A

PaxillinP-CadherinYB-1KRASc-Metp53BP1YAPSMAD4Cyclin E1Notch1p-AKT(S473)FibronectinSrcp-c-Met (Y1235)Beclin

c-

alpha-catenin14-3-3 epsilonp27HER2Claudin-7p21IGFBP2E-CadherinFOXO3ap-AKT(T308)SmacPTCHBIMN-CadherinBcl-XBcl-xLp-EGFR(Y992)VASPCDK4PCNAp70S6KChk2eEF2Kc-RAFSTAT5aMEK1SNAIlp-4E-BP1(T70)4E-BP1MSH6TIGARARp-YB-1(S102)FAKp-4E-BP1(S65)TTF1p-S6(S240/S244)p-4E-BP1(T37/T46)p-S6(S235/236)p-Rb(S807/S811)Cyclin B1CDK1INPP4BChk1p-p90RSK(T359/S363)p-EGFR(Y1068)JNK2VEGFR2Rad51p-Chk1(S345)c-IAPIGF-1R-beta

miR135a-5pNT

miR31NT

p-YAP (S12)

CDK1c-Myc

p-EGFR(Y1068)

Cyclin B1

JNK2

NF2 p-Chk1 (S345)

Cl PARP Src

Chk2STAT5A

YAPMSH6

p-4EBP1(T37/T46)

p-4EBP1 (S65)pRb(S807/811)ARp-HER3 (Y1298)p-4EBP1 (T70)VASPNotch1TTF1INPP4BSmacFOXO3a

p-mTOR(S2448)

PTCHmTOR

IGF-1R-betac-Metp-PKCA(S657)PKCAKRAS

HER3Smad3Cyclin E1Rab11p27Bcl-xLStathminp-HER2(Y1248)eEF2

B C

Suppl Fig 4

miR 1NT

Smacp-NFkBp65(S536)p-p38(T180Y182)BIMIGFBP2Bcl-XRab11Alpha-cateninKRASp27Cyclin E1HER2eEF2Kp-p27(T198)MEK1E-CadherinClaudin-7p53BP1mTORACC1Beta-cateninCaspase 7PKC-alphaeEF2p-EGFR(Y1068)TIGARc-RAFp-Rb(S807/S811)Cyclin B1p-p90RSK(T359/S363)TTF1p-Chk1(S345)CDK4p-4E-BP1(T70)Rad51p-AMPK(T172)p-AKT(T308)p-YAP(S127)CDK1Notch1ARp-AKT(S473)p-4E-BP1(S65)c-MycNotch3Fibronectinp-4E-BP1(T37/T46)IGF-1R-betaSrcCyclin D1p-c-Jun(S73)Smad1p21PCNAChk2VASPDvl3Gab2SRC3

miR221-5pNTD E Suppl Fig 4

TTF1Dvl3Gab2p-c-Jun(S73)Chk2INPP4Bp-EGFR(Y1068)SRC3STAT5-alphaJNK2Smad1p-p90RSK(T359/S363)p-Chk1(S345)p-Rb(S807/S811)p38 MAPKp-GSK alpha-beta(S21/S9)p70S6Kp-p38(T180/Y182)

Cyclin B1p-GSK3(S9)

FibronectinSrcp-AKT(S473)p-4E-BP1(S65)AKTp-YAP(S127)VEGFR2ARp-AKT(T308)E-CadherinCaspase-3SmacRab11c-KITmTORBeta-cateninYB-1Caspase-7KRASClaudin-7IGFBP2p-YB-1(S102)p53BP1Collagen VIp-c-Met(Y1235)p-p27(T198)HER3Alpha-cateninPKC-alphaMIG-6p-EGFR(Y992)Notch1HER2B

miR 133aNT

miR 133bNT

F G Suppl Fig 4

miR 143-3pNT

miR 24-1-3pNTIH Suppl Fig 4

miR 145-3pNT

miR 205NTJ K Suppl Fig 4

miR 221-3pNT

miR 222-3pNT

L M Suppl Fig 4


A

B

* p<0.05

*

*

* p<0.05

Supplemental Figure 6: Correlation w/ other Signatures

A

r =0.46p< 0.01

hsa-

miR

-31

si-AR (A) signature score

r =0.46p< 0.01

hsa-

miR

-133

asi-AR (A) signature score

r =0.46p< 0.01

hsa-

miR

-133

b


r =-0.02p= 0.86

hsa-

miR

-135

a-5p


r =0.35p< 0.01

hsa-

miR

-143

-5p


r =0.40p< 0.01

hsa-

miR

-145

-3p


r =0.27p= 0.01

hsa-

miR

-1


r =0.23P=0.03

hsa-

miR

-204


r =0.45p< 0.01

hsa-

miR

-205


r =0.34p< 0.01

hsa-

miR

-221

-5p


r =0.34p<0.01hs

a-m

iR-2

21-3

p


r =0.41p< 0.01hs

a-m

iR-2

22-3

p


Suppl Fig 6B

r =0.56p< 0.01

hsa-

miR

-133

asi-AR (B) signature score

r =0.56p< 0.01

hsa-

miR

-133

b

si-AR (B) signature score

r = 0.09p= 0.40hs

a-m

iR-1

35a-

5p


r = 0.47p= <0.01hs

a-m

iR-1

43-5

p


r = 0.51p< 0.01hs

a-m

iR-1

45a-

3p


r = 0.39p< 0.01

hsa-

miR

-1


r = 0.34p< 0.01

hsa-

miR

-204


r = 0.43p< 0.01

hsa-

miR

-205


r = 0.46p< 0.01

hsa-

miR

-221

-5p


r = 0.50p< 0.01

hsa-

miR

-221

-3p

r = 0.47p< 0.01

hsa-

miR

-222

-3p

si-AR (B) signature scoresi-AR (B) signature score

r =0.42p< 0.01

hsa-

miR

-133

a


Suppl Fig 6C

Genes induced by

miR135a

Genes suppressed by

miR135a

Suppl Fig 6D

Correlation of miR135a-induced gene expression changes with changes induced by AR (detected by using AR siRNAs A and B)

NES=-1.94 q<0.0181

Genes upregulated by AR

(downregulated by si-AR-A)

Enr

ichm

ent S

core

NES=-2.54 q<0.0001

Genes upregulated by AR

(downregulated by si-AR-B)

Enr

ichm

ent S

core

Genes induced by

miR135a

Genes suppressed by

miR135a


NES=-1.8821q<0.0888

Chandran Metastasis Up

Enric

hmen

t Sco

re

Genes induced by miR-

135a-5p

Genes suppressed by miR-

135a-5p

MIB miR133A1 miR1-2

PrEC

LNCaP

LNCaP

PrEC H3K4Me3

H3K27Me3

H3K4Me3

H3K27Me3

(0-50)

(0-50)

(0-50)

(0-50)

153 kb


A

B

C

PrEC

LNCaP

LNCaP

PrECH3K4Me3

H3K27Me3

H3K4Me3

H3K27Me3

(0-70)

(0-70)

(0-30)

(0-30)

miR-221miR-222

27 kb

miR31HGmiR-31

H3K4Me3

H3K27Me3

H3K4Me3

H3K27Me3

PrEC

LNCaP

LNCaP

PrEC (0-85)

(0-85)

(0-50)

(0-50)

85 kb


B

C

A

Supplemental Table 3. Evaluation of direct SiM-miRNA effects on the proteomic changes assayed via RPPA

Supplemental Table 2. Proteomic signature for 12 SiM-miRNAs transfected into LNCaP cells.

Supplemental Table 4. Prediction of SiM-miRNAs on steroid receptor coactivators.

Note: Using a union of five leading prediction algorithms: TargetScan, miRanda, PicTar, DianaLab, and miRDB, we queried whether the SiM-miRNAs could target p160 coactivators SRC1, SRC2 and SRC3. Table shows that SRC1 is predicted by 3 independent algorithms to be targeted by miR-135a-5p.

Supplemental Table 5. AGO HITS-CLIP and AGO PAR-CLIP analysis.

miRNA Site ReadsH3K4me3

PrEC

ReadsH3K4me3

LNCaP

P-value ReadsH3K27me

3 PrEC

ReadsH3K27me3 LNCaP

P-value

miR-135a-5p

chr1297856241-97859472

99/651 32/655 1.274e-08 45/663 98/843 0.003536

miR-221-5p

chrX45625956-45631024

486/785 32/392 4.203e-38 7/235 8/477 0.277

miR-1 chr1819319997-19322746

209/840 91/436 0.221 5/484 26/736 0.0082

miR-31 chr921554820-21560351

1234/2461

43/722 1.752e-67 26/392 188/1263 7.860e-05

Supplemental Table 6. Comparison of H3K4Me3 and H3K27Me3 signal at the miR-135a-5p, miR-221-5p, miR-1, and miR-31 loci in PrEC and LNCaP cells

supplemental table 1. oligonucleotide sequences used for mrna expression analysis by qpcr. primer...

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