0

1000

2000

3000

4000

0 10 20 30 40 50 60

EstramustineADT Dexamethasone

(Months)

(ng/mL)

PSA

Time

0

1000

2000

3000

4000

5000

0 20 40 60 80 100 120

ADT, Estramustine, Dexamethasone

PrednisoloneRadiotherapy

(Months)

(ng/mL)

PSA

Time

0

1000

2000

3000

4000

5000

0 20 40 60 80 100(Months)

(ng/mL)

PSA

Time

Estramustine

ADT

Dexamethasone

Radiotherapy

Docetaxel

Patient A

Patient B

Patient C

Supplementary Figure 1

Supplementary Figure 1. The clinical courses of three patients with CRPC.Treatments and PSA changes in three patients with CRPC. All patients were treated with ADT, estramustine, and dexamethasone. Furthermore, patients B and C underwent radiotherapy, and patient C was treated with docetaxel.

miR-222

miR-221

Supplementary Figure 2

Human chromosome Xp11.3 region

CTTCTTTTTTCTTCCACAGAGCCCCTCCCCAGAAGGCAAAGGATCACCCAGCTGCTGGAAGGTGTAGGTACCCTCAATGGCTCAGTAGCCAGTGTAGATCCTGTCTTTCGTAATCAGCAGCTACATCTGGCTACTGGGTCTCTGATGGCATCTTCTAGCTTCTGCTTCAGTGTCCTGAGAGAGATTATTGGGCAGTCTTTAATAATGAAAACTTTACCTTGAATTACACACACACACACACACACACACACACACACACACTCACACACACACACAAAGAGAGAGAGAGAGAGAGAGAGACAGAGAGAGAGGAGAGAGTCAAGTTCTAGGGTGTTAAGTCCAACATTATCAGCTGGGGCTTGGGAACCTTCAAGGTTTTATGAATGATGAGGTCTATTTGCTTTCAATACTACAAGGGGGAGGAATCTGGATATTTATCCATGTTTTACCTCTTACCTGTTTTTGTTTTACCTCTTTTTTTCTCTTTCCCCCCTATGAAAGGTATCATTTGGATAGATCAATCTATTCATCTATCTACCTACCTACCTATCTACCCCTACCCATCCACCCATTTATCCATCCACCCATCCATCCATCCATCCACCCACCTGCCCACGTACCTACCAGTTTATCTATCCGACCTTCCTTCCATCCAGCTTTTTCATCTCTTGACTTTTAGATGGCATTTTCAACATGATGTCATGATTAAATGATGACAACTTGATGATATGATGGTTTAAGGAAATTTTGTTGGTAGTAGGTAAGTCCCAGCATTTCTGACTGTTGGTTTTCTTTTCCTTGTGGTAGAAAAGACAAATGCCAATTTTGAAATCTTGAATGCAGTAGGCAGTTGTGTTGAAATAGTATGTGAGAATTACTTGCAAGCTGAACATCCAGGTCTGGGGCATGAACCTGGCATACAATGTAGATTTCTGTGTTCGTTAGGCAACAGCTACATTGTCTGCTGGGTTTCAGGCTACCTGGAAACATGTTCTCCATTGGCTGTCTCACCAATGCTACCCCTATAATGTTTCTGAATAACTGTT

44.5Mbp

miR-221miR-222

KRBOX4LOC392452

46.5Mbp46.0M45.0M 45.5M

KDM6A

CXorf36

ZNF674LINC01204

DUSP21

LINC01186

Supplementary Figure 2. The chromosomal location of miR-221/222 in the human genome. miR-221 and miR-222 are located close together on human chromosome Xp11.3 within 800 base pairs. The red characters show pre-microRNAs of miR-221 and miR-222. The underlines show mature miR-221 and miR-222.

Supplementary Figure 3

Patient A Patient B

Patient C

AR

H&E

No. 3 (Bone) No. 4 (Bone) No. 5 (Lung)

No. 6 (Liver) No. 7 (Dura) No. 8 (Bone)

AR

H&E

AR

H&E

Supplementary Figure 3. H&E staining and immunohistochemical staining of AR, PSA in the CRPC specimens used in array analysis.H&E staining was indicative of prostate cancer metastasis, and immunohistochemical staining for detection of AR showed heterogeneous expression of AR in CRPC specimens. PSA expression was high in all specimens.

PSA PSA

PSA

Supplementary Figure 4

C

0

20

40

60

80

100

CRPC-free rate

P = 0.0309

cM0 (n = 19)

cM1 (n = 33)

0 10 20 30 40 50 60 (Months)

D

0

20

40

60

80

100

CRPC-free rate

P = 0.848

PSA < 228 ng/mL(n = 26)

PSA > 228 ng/mL(n = 26)

0 10 20 30 40 50 60

A

0

20

40

60

80

100CRPC-free rate

P = 0.0403

cT2 or cT3(n = 33)

cT4 (n = 19)

0 10 20 30 40 50 60 (Months)

B

0

20

40

60

80

100

CRPC-free rate

P = 0.0548

cN0 (n = 13)

cN1 (n = 39)

0 10 20 30 40 50 60 (Months)

(%)

(%)

(%)

(%)

Time

Time Time

(Months)Time

Supplementary Figure 4. Associations between clinicopathological parameters and CRPC-free interval.Kaplan-Meier survival curves for CRPC progression-free survival compared to (A) cT stage, (B) cN stage, (C) cM stage and (D) serum PSA. P-values were calculated using the log-rank test.

TargetScan Database(release 19.0)2,275 genes

GEO DatabasePCa expression data

(GSE 29079)PCa upregulated genes(fold change > 0.5)

135 genes

Gene expression analysismiR-221/222 transfectioninto PC3 and DU145 cells

(fold change < -0.1)

Supplementary Figure 5

Putative target genes

Supplementary Figure 5. Flow chart of the strategy for analysis of miR-221/222 cluster target genes.A total of 2275 genes were selected as putative miR-221/222 target genes by TargetScan database analysis. We then analysed the expression levels of these candidate genes by using available data sets in the GEO (GSE 29079). The analyses showed that 135 genes were significantly upregulated in PCa specimens compared with normal specimens. Finally, gene expression analysis data of miR-221- and miR-222-transfected PC3 and DU145 cells were merged.

56.11 54.5648.47

57.37

11.85 14.9316.52

13.62

32.05 30.51 35.0129.00

0

10

20

30

40

50

60

70

80

90

100

mock control miR-221 miR-222

G0/G1 S G2/M

(%)G0/G1 S G2/M

Cell counts

mock

G0/G1 S G2/M

Cell counts

control

G0/G1 S G2/M

Cell counts

miR-221

G0/G1 S G2/M

Cell counts

miR-222

% of cell counts

A

B

Supplementary Figure 6. Effects of miR-221/222 transfection on cell cycle in PC3.(A) The bar charts represent the percentage of the cells in the G0/G1, S, or G2/M phase. (B) Typical results of cell cycle analysis of mock, miR-control, miR-221, or miR-222 transfectants in PC3.

Supplementary Figure 6

mock

PI

Dead Apoptotic

Earlyapoptotic

control

PI

Dead Apoptotic

Earlyapoptotic

miR-221

PI

Dead Apoptotic

Earlyapoptotic

miR-222PI

Dead Apoptotic

Earlyapoptotic

0

20

40

60

80

100

120

Apoptotic cells

(relative to mock)

(%)

mock

control

miR-221

miR-222

Supplementary Figure 7. Effects of miR-221/222 transfection on apoptosis in PC3.Ratio of the apoptotic cell fractions (early apoptotic + apoptotic cells) in miR-221 or miR-222 transfectants in comparison with mock and miR-control transfectants.

Supplementary Figure 7

Supplementary Figure 8

BA C

250

150

10075

5037

25201510

kDa

250

150

10075

5037

25201510

kDa

DU145

mock

control

miR-221

miR-222

si-Ecm29

PC3

mock

control

miR-221

miR-222

PC3

mock

control

miR-221

miR-222

PC3

mock

control

miR-221

miR-222

anti-Ecm29 antibody 1:1000 anti-Ecm29 antibody 1:1000 anti-Ecm29 antibody 1:1000

anti-GAPDH antibody 1:2000anti-GAPDH antibody 1:2000anti-GAPDH antibody 1:2000

210kDa

si-Ecm29

PC3

mock

control

si-Ecm29

DU145

mock

control

250

150

10075

50

37

2520

kDa

250

150

10075

50

37

2520

210kDa210kDa

kDa

250150

10075

50

37

2520

1510

kDa

36kDa36kDa

36kDa

250150

10075

50

37

2520

1510

kDa

Supplementary Figure 8. Western blotting data of anti-Ecm29 antibody and anti-GAPDH antibody.The molecule weight of Ecm29 is 210kDa and GAPDH is 36kDa.

Supplementary Figure 9

Supplementary Figure 9. Ecm29 expression in other cancer tissues.Ecm29 expressions were relatively high in muscle invasive bladder cancer and pancreatic cancer compared with normal tissues (GSE15471 and GSE3167).

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

P = 0.0127

Normal(n = 39)

Pancreaticcancer

(n = 39)

20

40

60

80

100

120

140

160

180

Expression of Ecm29

Normal(n = 9)

Muscle invasive Bladder cancer

(n = 13)

P = 0.0045

Expression of Ecm29