MIRNAS ARRAYS: DATABASES AND PLATFORMS UBio Training Courses Gonzalo Gómez//ggomez@cnio.es

miRNAs Firstly detected in C. elegans (V. Ambros, 1993) Mapping to non-coding regions (introns) Pri-miRNA processed by Drosha DICER removes the structural loop mature miRNA: ssRNA, 22 nucleotides miRNA-RISK complex: mRNAs post-transcriptional inhibition 33% of human genes are supposed to be regulated by miRNAs.

miRNAs Biological role Brain development (miR-430) • Nervous system development(miR-273) • Pancreatic Langerhans islands development (miR-375) • Adipocytes development (miR-143) • Heart development(miR-1) Inmune response (miR-223, cluster miR-17~92, miR-146a,miR-155…) • Apoptosis (miR-14) Maduracion linea mieloide y linfoide

miRNAs and cancer OncomiRs  Proliferation  Invasion Oncogene  Angiogenesis  Cell death Oncogene miRNA Proliferation Invasion Angiogenesis  Cell death Tumor suppressor Upregulation Tumor formation Downregulation miR-148a y miR-34b/c parecen inhibir la diseminacion tumoral regulando genes relacionados con la metastasis. Cuando estan los genes de los miRs estan hipermetilados se upregulan los genes de metástasis genes favoreciendo la diseminacion. Esquela-Kerscher & Slack. Nature Reviews Cancer. 2006.

miRNAs Target sites miRNAs seed region: 5’, nucleotides 2-7(8) Most gene targets of a given miRNA have only a single 7 nt matching to that miRNA seed region. 7-8 nt hundreds of target predictions for each miRNA family (~300 conserved targets per miRNA family in vertebrates) High rate of false positives in predictions. Bartel D. Cell 2009.

Target Prediction Algorithms miRNAs Target Prediction Algorithms PREDICTION CRITERIA miR seed-target complete base-pairing Interspecies conservation Number of binding sites for a given 3’UTR in a particular gene Free energy for the miR-target duplex Binding site accessibility miRNA- target secondary structure Overlapping entre los metodos (targetSacn, PicTAR, EMBL) es bastante alto aunque no al 100% . Sources of these differences could be: alignment artifacts, use of slightly different UTR DBs, use of different miRNAs sequences… Other prediction algorithms… Bartel D. Cell 2009. More target prediction tools: http://en.wikipedia.org/wiki/List_of_RNA_structure_prediction_software#Inter_molecular_interactions:_MicroRNA:UTR

miRBase miRNAs Databases H. Sapiens ~695 miRNAs Version Date miRNAs 1.0 12/02 218 1.1 01/03 262 1.2 04/03 295 1.3 05/03 332 1.4 07/03 345 2.0 07/03 506 2.1 09/03 558 2.2 11/03 593 3.0 01/04 719 3.1 04/04 899 4.0 07/04 1185 5.0 09/04 1345 5.1 12/04 1420 6.0 04/05 1650 7.0 06/05 2909 7.1 10/05 3424 8.0 02/06 3518 8.1 05/06 3963 8.2 07/06 4039 9.0 10/06 4361 9.1 02/07 4449 9.2 05/07 4584 10.0 08/07 5071 10.1 12/07 5395 11.0 04/08 6396 12.0 09/08 8619 13.0 03/09 9539 14.0 09/09 10833 http://www.mirbase.org/ H. Sapiens ~695 miRNAs M. musculus: ~488 miRNAs

miRNA nomenclature (miRBase) miRNAs miRNA nomenclature (miRBase) mir: immature sequence (hairpin). E.g. hsa-mir-203 b) miR: mature miRNA sequence. E.g. hsa-miR-203 - a/b: paralog miRs, difer in 1-2 nucleotides. E.g. hsa-miR-9a, hsa-miR-9b - 1-2: Identical miRs, different hairpin. Ej. hsa-miR-19b-1, hsa-miR-19b-2 - 5p-3p: mature miR generated from precursor 5´ (or 3) sequence. E.g. hsa-miR-17-5p - *: Minor transcript complementary to mature miR. E.g. hsa-miR-33a* Paralogos: origen por duplicacion intraespecie

TarBase miRNAs Databases http://microrna.gr/tarbase Contains only those miRNA-target relationships experimentally validated TarBase: Contiene solo miRNAs-targets verificados experimentalmente. Logicamente tiene menios información. The database of experimentally supported targets: a functional update of TarBase. Papadopoulos GL, Reczko M, Simossis VA, Sethupathy P, Hatzigeorgiou AG., Nucleic Acids Res. 2009 Jan;37(Database issue):D155-8.

miRNAs and disease Databases http://www.mir2disease.org/ http://cmbi.bjmu.edu.cn/hmdd Cancer (Calin and Croce 2006; Ura et al 2008; Stamatopoulos et al 2009…) Cardiovascular disease (Latronico et al. 2007; van Rooij and Olson 2007) Schizophrenia (Hansen, et al. 2007; Perkins et al. 2007) Renal misfunction (Williams 2007) Tourette syndrome (Esau and Monia 2007) Psoriasis (Sonkoly et al. 2007) Muscle disorders (Eisenberg et al. 2007), X fragile syndrome (Fiore and Schratt 2007) Policitemia vera (Bruchova et al. 2007) Diabetes (Williams 2007) Chronic hepatitis (Murakami et al. 2006) AIDS (Hariharan etal. 2005) Obesity (Weiler et al. 2006, Lovis et al. 2008, Xie et al. 2009).

miRNAs Detection Methods

miRNA microarrays Commercial platforms Human, rat, mouse dog, chimpanzee, etc EXIQON LNA = Locked Nucleic Acid LNA es una modificacion quimica que consiste en: ribose ring is "locked" with a methylene bridge connecting the 2’-O atom and the 4’-C atom. LNA hace mas estable el par DNA-miR para perfect match y 1mismatch. INVITROGEN: Human, rat, mouse Human, multispecie

Agilent recommendations. No normalization miRNA microarrays Commercial platforms Agilent recommendations. No normalization Normalization 75th percentile (75th value = 1) Abutting: colindando. Algunos autores (Pedro Lopez-Romero) proponen RMA with no background correction. Additional G-C pair in the probe-target interaction region stabilizes targeted miRNAs relative to homologous RNAs. Additionally, all probes contain a 5' hairpin (blue), abutting the probe-target region, to increase target and size miRNA specificity

miRNA microarrays Commercial platforms LNA = Locked Nucleic Acid Preprocessing scripts provided by Exiqon Background correction = normexp Normalization = quantiles RG_normexp <- backgroundCorrect(RGfilt_0, method = "normexp", offset=50) MA_norm <- normalizeBetweenArrays(RG_normexp$G, method="quantile") MA_lognorm<-log2(MA_norm) EXIQON LNA = Locked Nucleic Acid LNA es una modificacion quimica que consiste en: ribose ring is "locked" with a methylene bridge connecting the 2’-O atom and the 4’-C atom. LNA hace mas estable el par DNA-miR para perfect match y 1mismatch. LNA is chemical modification. Ribose ring is "locked" with a methylene bridge connecting the 2’-O atom and the 4’-C atom. LNA makes DNA-miR pairs more stable (higher Tm) when perfect match and 1mismatch hybridizations occurs.

OK! 20 normalized arrays 600 miRNAs 2 classes (healthy y tumor) miRNA microarrays Differential expression analysis 20 normalized arrays 600 miRNAs 2 classes (healthy y tumor) T test, SAM, limma FWER FDR METHOD Pvalue adjustment GEPAS Asterias SAM tools… pvalue OK! Differentially expressed miRNAs between classes FWER: Type I Family Wise Error Rate FDR: False Discovery Rate

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