ADT, Estramustine, Dexamethasone Prednisolone Radiotherapy (Months) (ng/mL) PSA Time 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 CTTCTTTTTTCTTCCACAGAGCCCCTCCCCAGAAGGCAAAGGATCACCCAGCTGCTGGAAGGTGTAG GTACCCTCAATGGCTCAGTAGCCAGTGTAGATCCTGTCTTTCGTAATCAGCAGCTACATCTGGCTAC TGGGTCTCTGATGGCATCTTCTAGCTTCTGCTTCAGTGTCCTGAGAGAGATTATTGGGCAGTCTTTA ATAATGAAAACTTTACCTTGAATTACACACACACACACACACACACACACACACACACACTCACACA CACACACAAAGAGAGAGAGAGAGAGAGAGAGACAGAGAGAGAGGAGAGAGTCAAGTTCTAGGGTGTT AAGTCCAACATTATCAGCTGGGGCTTGGGAACCTTCAAGGTTTTATGAATGATGAGGTCTATTTGCT TTCAATACTACAAGGGGGAGGAATCTGGATATTTATCCATGTTTTACCTCTTACCTGTTTTTGTTTT ACCTCTTTTTTTCTCTTTCCCCCCTATGAAAGGTATCATTTGGATAGATCAATCTATTCATCTATCT ACCTACCTACCTATCTACCCCTACCCATCCACCCATTTATCCATCCACCCATCCATCCATCCATCCA CCCACCTGCCCACGTACCTACCAGTTTATCTATCCGACCTTCCTTCCATCCAGCTTTTTCATCTCTT GACTTTTAGATGGCATTTTCAACATGATGTCATGATTAAATGATGACAACTTGATGATATGATGGTT TAAGGAAATTTTGTTGGTAGTAGGTAAGTCCCAGCATTTCTGACTGTTGGTTTTCTTTTCCTTGTGG TAGAAAAGACAAATGCCAATTTTGAAATCTTGAATGCAGTAGGCAGTTGTGTTGAAATAGTATGTGA GAATTACTTGCAAGCTGAACATCCAGGTCTGGGGCATGAACCTGGCATACAATGTAGATTTCTGTGT TCGTTAGGCAACAGCTACATTGTCTGCTGGGTTTCAGGCTACCTGGAAACATGTTCTCCATTGGCTG TCTCACCAATGCTACCCCTATAATGTTTCTGAATAACTGTT 44.5Mbp miR-221 miR-222 KRBOX4LOC Mbp 46.0M45.0M45.5M 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

Supplementary Figure 4 C CRPC-free rate P = cM0 (n = 19) cM1 (n = 33) (Months) D CRPC-free rate P = PSA < 228 ng/mL (n = 26) PSA > 228 ng/mL (n = 26) A CRPC-free rate P = cT2 or cT3 (n = 33) cT4 (n = 19) (Months) B CRPC-free rate P = cN0 (n = 13) cN1 (n = 39) (Months) (%) 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 Database PCa expression data (GSE 29079) PCa upregulated genes (fold change > 0.5) 135 genes Gene expression analysis miR-221/222 transfection into 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.

mockcontrolmiR-221miR-222 G0/G1SG2/M (%) G0/G1SG2/M Cell counts mock G0/G1SG2/M Cell counts control G0/G1SG2/M Cell counts miR-221 G0/G1SG2/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 Early apoptotic control PI Dead Apoptotic Early apoptotic miR-221 PI Dead Apoptotic Early apoptotic miR-222 PI Dead Apoptotic Early apoptotic Apoptotic cells (relative to mock) (%) mock controlmiR-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 BAC kDa 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-GAPDH antibody 1: kDa si-Ecm29 PC3 mock control si-Ecm29 DU145 mock control kDa kDa kDa kDa 36kDa 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) P = Normal (n = 39) Pancreatic cancer (n = 39) Expression of Ecm29 Normal (n = 9) Muscle invasive Bladder cancer (n = 13) P = Expression of Ecm29