1 SUPPLEMENTAL FILE 2 3 mir-22 and mir-29a are members of the androgen receptor cistrome modulating LAMC1 and Mcl-1 in prostate cancer 4 5 6 Lorenza Pasqualini 1, Huajie Bu 1,2, Martin Puhr 1, Narisu Narisu 3, Johannes Rainer 4, Bettina Schlick 1,5, Georg Schäfer 1,6, Mihaela Angelova 7, Zlatko Trajanoski 7, Stefan T. Börno 8, Michal R. Schweiger 8,9, Christian Fuchsberger 10, Helmut Klocker 1 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1 Department of Urology, Division of Experimental Urology, Medical University of Innsbruck, Innsbruck, Austria 2 Research Institute for Biomedical Aging Research, University of Innsbruck, Innsbruck, Austria 3 Medical Genomics and Metabolic Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA 4 Biocenter Innsbruck, Section for Molecular Pathophysiology, Medical University of Innsbruck, Innsbruck, Austria. 5 Oncotyrol, Center for Personalized Cancer Medicine, Innsbruck, Austria 6 Department of Pathology, Medical University of Innsbruck, Innsbruck, Austria 7 Biocenter Innsbruck, Division of Bioinformatics, Medical University of Innsbruck, Innsbruck, Austria. 8 Max Planck Institute for Molecular Genetics, Berlin, Germany 9 Cologne Center for Genomics, University of Cologne, Germany; 10 Department of Biostatistic,, University of Michigan, Ann Arbor, USA Abbreviated Titel: Androgen regulation of mir-22 and mir-29a 25 Pasqualini et al. Androgen regulation of mir-22 and mir-29a Supplement 1
26 27 28 Supplemental Table 1 Predicted androgen responsive elements (AREs) within the androgen receptor binding sites (ARBSs) of mir-22, mir29a and mir-17-92 cluster ARBSs Detailed Matrix Information Number of predicted AREs Matrix similarity Predicted Sequence mir-22 ARBS_1 AR binding site 3 0.937 tcggggctttctgtcctca mir-22 ARBS_2 AR binding site 1 0.963 tgggaacagcttgtgctgc mir-22 ARBS_3 AR binding site 1 0.928 acagagctttctgtccctg mir-29a ARBS_1 AR binding site 5 0.869 ttgggccttagtgttctca mir-17-92 cluster ARBS_2 AR binding site 2 0.906 taagaactctgggttctcc 29 30 31 32 33 34 35 Detection of androgen responsive elements in the ChIP-seq-identified ARBSs of mir-22, mir-29a and mir-17-92 cluster using MatInspector software. A matrix similarity score of 1.00 indicates a perfect match to the matrix, implying that the candidate ARBS genomic sequences include well conserved AREs. Additionally, the highly conserved base pairs are marked in bold and the ones in capital letters represent the core sequence used by MatInspector. 36 Pasqualini et al. Androgen regulation of mir-22 and mir-29a Supplement 2
37 38 39 40 41 42 43 44 45 Supplemental Figure 1 Schematic depiction of the genomic organization of mir-22, mir-29a and mir-17-92 cluster sites. Indicated are the genomic position of mir-22, mir-29a and the mir-17-92 cluster along with ARBSs and the adjacent genes localization according to UCSC Genome Browser on human Feb. 2009 (GRCh37/hg19) assembly. Red triangles represent ARBSs, their size reflects the extent of AR enrichment for each binding site according to ChIP-seq sequence reads. For the mir-17-92 cluster, triangles with the same color represent mirnas which share a similar sequence. 46 Pasqualini et al. Androgen regulation of mir-22 and mir-29a Supplement 3
47 48 49 50 51 52 53 54 55 56 57 58 59 60 Supplemental Fig.2 Androgen regulation of mir-22 and mir-29a in PC3-AR cells (A) Relative expression of androgen receptor protein in PC3-AR cells in comparison with its parental AR negative cell line PC3 and the AR positive LNCaP cells, used as positive control. Protein values were normalized to GAPDH and quantified using the Odyssey IR Imaging System. (B) Real-time PCR analysis of androgen induced up-regulation of mir-22 and MIR22HG in PC3-AR cells. This cell line was established by stable overexpression of wild-type AR in the AR negative PC3 cell line. Cells were treated either with 1nM R1881 or equivalent vehicle (Ctrl) for 24 and 48 h before mrna/mirnas isolation. mirna expression was normalized to the small nucleolar RNA, SNORD44. FKBP5 was utilized as positive control and TBP was chosen as normalizer for AR and FKBP5 gene expression. Each bar represents mean values + SEM of at least three independent experiments, (Unpaired Student st-test, * P <0.05; ** P<0.01; *** P<0.001). 61 Pasqualini et al. Androgen regulation of mir-22 and mir-29a Supplement 4
62 63 64 65 66 67 68 69 70 Supplemental Figure 3 mir-22 and mir-29a levels in malignant and non-malignant prostate tissue based on mirna-seq data extracted from The Cancer Genome Atlas (TCGA). (A) Comparison of mirna expression levels in 52 matched cancer-benign tissue samples obtained from primary tumors. DESeq statistical tool was used and log 2 values are shown in the graph, (* P <0.05; ** P<0.01; *** P<0.001). (B) A total of 324 patients were classified into subgroups according to the Gleason score (low 6, medium =7, high >7) and the different expression levels of mir-22 and mir-29a were evaluated by means of ANOVA statistical test, (* P <0.05; ** P<0.01; *** P<0.001). 71 Pasqualini et al. Androgen regulation of mir-22 and mir-29a Supplement 5
72 73 74 75 76 77 78 79 80 81 82 83 84 Supplemental Figure 4 (A) mir-22 and mir-29a levels in LNCaP and DUCaP cells transfected for 48 h with mir-22 or mir-29a mimics were measured by real-time PCR. mirnas expression was normalized to the small nucleolar RNA, SNORD44 and levels are depicted in a log 10 scale. (B) Representative cell culture images of mir-22/mir-29a mimics treated DUCaP and LNCaP cells. Rounding and detachment of cells is recognizable, in particular after treatment with mir-22 due to the expected downregulation of the basement membrane protein LAMC1. (C) Relative expression of LAMC1 protein in DUCaP cells transfected with mir-22 or mir- 29a mimics for 48 h. Protein content was quantified using the Odyssey IR Imaging System and normalized to GAPDH. Values are expressed relative to the mimic Ctrl and each bar represents the mean value + SEM of at least three independent experiments, (Unpaired Student s T-test, * P <0.05; ** P<0.01; *** P<0.001). 85 Pasqualini et al. Androgen regulation of mir-22 and mir-29a Supplement 6
86 87 88 89 90 91 92 93 94 Supplemental Figure 5 Differential DNA methylation or AR target genomic loci in cell lines. MeDIP-seq data obtained for a panel of prostate cancer cell lines was analyzed for the differential DNA methylation of mir-22 and mir-29a genomic loci encompassing the ARBSs. Depicted are the MeDIP-seq values (rpm). The plots show an elevated DNA methylation for the AR positive cell lines LNCaP and VCaP having low basal levels of both mirnas and a minimal DNA methylation in the AR negative cell lines PC3 and DU145 displaying high basal mirnas expression. MeDIP-seq values of DNA derived from benign and malignant prostate tissue samples are included for comparison. Pasqualini et al. Androgen regulation of mir-22 and mir-29a Supplement 7