1. Miki Fuse 1, Satoko Kojima 2, Takeshi Chiyomaru 3, Hideki Enokida 3, Kazumori Kawakami 3, Hirofumi Yoshino 3, Nijiro Nohata 1, Yukio Naya 2, Tomohiko Ichikawa 4, Masayuki Nakagawa 3, Naohiko Seki 1 1 Department of Functional Genomics, Graduate School of Medicine, Chiba University,, 2 Depatrment of Urology, Teikyo University Chiba Medical Center, 3 Department of Urology, Graduate School of Medicine and Dental Sciences, Kagoshima University, 4 Department of Urology, Chiba University Graduate School of Medicine Objectives : Our recent analyses of miRNA expression signatures showed significant reduction of microRNA-1 (miR-1) and microRNa-133a (miR-133a) in several types of cancer 1,2,3. miR-1 and miR-133a are located on the same chromosomal locus in the human genome. miR-1-1/miR-133a-2 and miR-1-2/miR-133a-1 are clustered on different chromosomal regions, 20q13.33 and 18q11.2, respectively. We examined the functional significance of miR-1 and miR-133a in prostate cancer (PCa) cells and identified the novel molecular targets regulated by both miR-1 and miR-133a. Molecular target regulated by tumor suppressive microRNA-1 and microRNA-133a in prostate cancer Conclusions: Down-regulation of miR-1 and miR-133a was a frequent event in PCa and both function as tumor suppressors. PNP is a novel target gene of miR-1 and miR-133a and potentially functions as an oncogene. Therefore, identification of novel molecular networks regulated by miRNAs may provide new insights into the underlying causes of PCa oncogenesis. References: 1, miR-145, miR-133a and miR-133b: Tumor-suppressive miRNAs target FSCN1 in esophageal squamous cell carcinoma. Kano et al, Int.J Can 2010 127:2804-2814 2, The tumour-suppressive function of miR-1 and miR-133a targeting TAGLN2 in bladder cancer. Yoshino et al, Br J Can 2011 104:808-818 3, Tumour suppressive microRNA-874 regulates novel cancer networks in maxillary sinus squamous cell carcinoma. Nohata et al, Br J Can 2011 105:833-841 Key findings: 1.The expression levels of miR-1 and miR-133a were significantly down-regulated in PCa compared to non-PCa tissue and each of expression levels were highly correlated. 2.Cell proliferation, migration and invasion were inhibited in of miR-1 or miR-133a transfectants in comparison with mock and the control transfectants. 3.Genome-wide gene expression analysis and luciferase reporter assay showed that purine nucleoside phosphorylase (PNP) was directly regulated by both miRNAs. 4.Silencing of the PNP gene inhibited proliferation, migration, and invasion in both PCa cell lines. 5.Immunohistochemistry demonstrated positive staining of PNP in PCa specimens. PIN showed weaker staining of PNP as well. Methods : 1.The expression levels of miR-1 and miR-133a in PCa clinical samples were examined by real-time PCR. 2.The function of miR-1 and miR-133a were examined in PCa cell line (PC3 and DU145) using XTT assay, invasion assay and migration assay. 3.Molecular targets were identified by genome-wide gene expression analysis and luciferase reporter assay. 4.To examine the functional role of PNP, we performed loss-of-function analysis using two different si-PNP transfections into PCa cell lines. 5.The expression levels of the target gene of miR-1 and miR-133a, Purine nucleoside phosphorylase (PNP) in PCa clinical samples were examined by RT-PCR and Immunohistochemistry. PC3 DU145 PC3 DU145 7. Repression of PNP by miR-1 and miR-133a X100X400 PCa (GS3+4 pT3N0) PIN Non-PCa PNP expression 9. Tissue microarray PNP express strongly in PCa, slightly in PIN Cell proliferation Wound healing assayInvasion assay 8. Knock-down of PNP induced inhibition of cell proliferation, migration, and invasion in PC3 and DU145 cells PC3 DU145 Cell proliferation (% of mock) 0 2020 4040 6060 8080 100 120 ** 5. Expression levels of possible target gens of miR-1 and miR-133a 6. miR-1 and miR-133a binding sites in 3’-UTR of PNP mRNA miR-1 expression miR-133a expression Correlation of miR-1 and miR133a expression 0 5 10 15 20 25 30 P<0.0001 miR-1 Non-PCa PCa expression levels of miRNA 0 2 4 6 8 10 12 14 16 18 P<0.0001 miR-133a Non-PCa PCa 2. Expression levels of miR-1 and miR-133a in PCa 1. Patients characteristics Characteristic Age (years) Median (range) PSA (ng/ml) Median (range) T stage T3a T3b T4 N stage N0 N1 M stage M0 M1 Gleason score 4+3 4+4 4+5 5+5 Non-PCa (n=22) PCa (n=28) 72 (63-88) 228 (7.23-2640) 8 (29%) 12 (42%) 8 (29%) 9 (32%) 19 (68%) 10 (36%) 18 (64%) 2 (7%) 14 (50%) 11 (39%) 1 (4%) 66 （ 53-72 ） 6.7 (4.3-19.5) 4. Down-regulated genes in miR-1 and miR-133a transfectants 8407 51776 79819 83990 5819 201895 4860 29956 79026 4323 53340 252983 56267 146779 TAGLN2 ZAK WDR78 BRIP1 PVRL2 C4orf34 PNP LASS2 AHNAK MMP14 SPA17 STXBP4 CCBL2 EFCAB3 -3.40 -1.77 -1.63 -2.95 -2.80 -2.33 -2.14 -1.15 -1.33 -1.21 -1.43 -1.03 -1.14 -1.10 -2.60 -2.97 -2.91 -1.86 -1.57 -2.28 -1.31 -2.45 -2.15 -1.01 -1.40 -1.22 -1.08 -1.56 -1.38 -1.22 -1.04 -1.78 -1.17 -2.08 -1.12 -1.23 -1.52 -1.42 -1.09 -1.14 -1.16 -1.71 -2.33 -1.81 -1.68 -1.06 -1.38 -1.54 -1.48 -1.23 -1.54 -1.11 -1.35 -1.02 -1.17 -2.32 -2.11 -1.89 -1.88 -1.80 -1.79 -1.77 -1.55 -1.48 -1.32 -1.24 -1.22 -1.13 +-+-++++---+--+-+-++++---+-- +-+--+++-+-++-+-+--+++-+-++- Entrez Gene ID Symbol miR-1 miR-133a miR-1 miR-133a miR-1 miR-133a Average PC3 DU145 Fold Change (log2 ratio) Target sites Cell proliferationWound healing assay Invasion assay miR-133a miR-1 control mock Cell proliferation (% of mock) 0 2020 4040 6060 8080 100 120 PC3DU145 ** * * 3. miR-1 and miR-133a inhibit cell proliferation, migration, and invasion in PC3 and DU145 cells 0 0.2 0.4 0.6 0.8 1.0 1.2 Position 365-371 deletion Position 407-413 deletion - + + - - - + + miR-133a miR-1 control Wound closure (% of mock) 0 2020 4040 6060 8080 100 120 PC3DU145 ** PC3 DU145 Invasion cell number (% of mock) 0 2020 4040 6060 8080 100 120 ** **: P < 0.0001 *: p < 0.0005 si-PNP-2 si-PNP-1 si-control mock PC3 DU145 Wound closure (% of mock) 0 2020 4040 6060 8080 100 120 ** PC3 DU145 Invasion cell number (% of mock) 0 2020 4040 6060 8080 100 120 ** **: P < 0.0001 11.. Abstract #3226