Recent Work & Current Methods in Cancer microRNA Research

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• Brief review of microRNA basics: history, biogenesis, function • Recent developments of microRNA research in the field of cancer genomics • Current methods for microRNA discovery and profiling • Case studies and application examples

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Cancer microRNA Webinar Recent Work & Current Methods In Cancer microRNA Research Christoph Eicken , PhD Head of Technical Services – Microarrays 2012-07-11

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Agenda Recent Studies Current methods Q & A Case Studies miRNA Intro

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Transcriptomics microRNA Profiling Microarray Services microRNA Discovery Sequencing Services Seq- Array SM Method Proteomics Phosphopeptide Binding Microarray Service Protein- Protein Interaction Microarray Service Kinase Profiling Microarray Service 5 µ Paraflo ® Microfluidic Technology High Density – On Chip Parallel Synthesis Customizable – DNA, RNA, Peptides, and Analogs Quality Data – Microfluidics / Synthesis Chemistry

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A ll miRNAs are small non-coding RNAs, usually consisting of ∼20–22 nucleotides for animals and ∼20–24 nt for plants. All miRNA precursors have a well-predicted stem loop hairpin structure, and this fold-back hairpin structure has a low free energy Many miRNAs are evolutionarily conserved, some from worm to human in animals, or from ferns to core eudicots or monocots in plants Bind to complementary mRNA molecules and act as negative regulators of translation High copy number Expression is tissue (and developmental stage) specific microRNAs - What W e Know

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microRNAs - What W e Know Currently - 21643 mature miRNAs across 168 plant, animal, and virus species ( miRBase 18 , Nov. 2011 ). Mechanism is far reaching and complex – each miRNA may control many genes and it is estimated that miRNAs regulate expression of up to 1/3 of all human genes. Operate by one of two hypothesized mechanisms : – Complete pairing mRNA is degraded - predominant in plants – Imperfect pairing translation is repressed but mRNA remains intact - predominant in animals

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1993 – Lin-4 was shown to encode two small RNA molecules (not protein) Small RNAs control developmental timing in C. elegans through negative regulation of lin-14 gene. Lee RC et al. 1993 The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75(5):843-54. [ article ] 1998 – RNAi is observed for the first time – leads to Nobel Prize (2006) Fire A, Mello CC et al. 1998 Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998; 391:806-811. [ article ] Challenge to the Central Dogma of Biology DNA > transcription > RNA > translation > protein 2000 – Let-7 was identified in humans and Drosophila . (Reinhart et al., Slack et al.) 2001 – Bartel , Tuschl , Ambros - Discover large class of small regulatory RNAs, name them microRNA (miRNA) Lau NC et al. 2001 An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans . Science 294(5543):858-62 . [ abstract ] Lagos-Quintana M et al. 2001 Identification of novel genes coding for small expressed RNAs . Science 294(5543):853-8 . [ abstract ] Lee RC et al. 2001 An extensive class of small RNAs in Caenorhabditis elegans . Science 294(5543):862-4. [ abstract ] 2002 – Identification of Drosha reveals complete microRNA biogenesis pathway pri-miRNAs > Drosha > pre- miRNAs (~70nt) > Dicer > mature miRNAs (~22nt) Lee Y et al. (2002) The nuclear RNase III Drosha initiates microRNA processing. Nature 425(6956):415-9. [ abstract ] Milestones

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2002 – Croce Lab - first report about the role of miRNAs in cancer Established that the gene cluster containing the miRNAs miR-15 and miR-16 is deleted in most people with chronic lymphocytic leukaemia (CLL). Calin GA, Dumitru CD, Shimizu M et al. (2002) Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. PNAS USA, 99:15524–15529. [ article ] Further studies have shown that miR-15 and miR-16 act as tumour suppressors by targeting the oncogene BCL2 , which encodes a protein involved in cell survival. 2003 – Stoffel Lab - First report of in vivo silencing of miRNAs with antagomirs used chemically engineered oligonucleotides Krutzfeldt J et al. (2005) Silencing of microRNAs in vivo with ' antagomirs '. Nature 438(7068):685-9. [ abstract ] 2004 – Microarrays are used to profile miRNA expression Several groups develop or modify existing gene expression microarrays for profiling miRNAs Babak et al. 2004 [ article ], Barad et al. 2004 [ article ], Liu et al. 2004 [ article ], Nelson et al. [ abstract ], 2004, Thomson et al. 2004 [ article ]. Milestones

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2005 – Three landmark studies published in Nature definitively link miRNAs with cancer development Lu et. al. were able to successfully classify poorly differentiated tumours using miRNA expression profiles. [ article ] He et al. demonstrated that non-coding RNAs, specifically microRNAs, can modulate tumour formation, and implicate the mir-17−92 cluster as a potential human oncogene . [ article ] O'Donnell et. al. showed that the c- Myc oncogene activates expression of a cluster of six miRNAs on human chromosome 13. [ abstract ] 2005 – Next-Gen Sequencing is used for small RNA discovery and analysis Green Lab - uses Solexa (now Illumina ) sequencing to identify novel small RNAs in Arabidopsis plants Lu C et al. 2005 Elucidation of the small RNA component of the transcriptome . Science, 309: 1567–1569 . [ abstract ] 2006 – miRBase goes online at Sanger Institute Depository for experimentally verified microRNAs: http://www.mirbase.org/ Griffiths-Jones S et al. 2006 miRBase: microRNA sequences, targets and gene nomenclature. NAR 34(Database Issue):D140-D144. [ article ] 2008 – Circulating miRNAs detected in body fluids miRNA signatures in serum and plasma provide cancer diagnosis / prognosis Serum - Chen X et al.(2008) Characterization of microRNAs in serum: A novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res 18:997–1006. [ article ] Plasma - Mitchell PS et al.(2008) Circulating microRNAs as stable blood-based markers for cancer detection. PNAS USA 105:10513–10518. [ article ] Milestones

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pri-miRNA = primary microRNA transcript pre- miRNA = precursor microRNA miRNA * = antisense microRNA (now -3p or -5p) miRISC = microRNA-induced silencing complex For latest information regarding miRNA nomenclature see the miRBase.org blog . miRNA Processing

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miRNA Biogenesis Pathway (A) Animal and (B) plant miRNA biogenesis. Mature miRNAs are indicated in red and miRNA* strands are in blue. Du T, Zamore PD. 2005 microPrimer : the Biogenesis and Function of microRNA. Development 132: 4645-4652. [ article ]

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miRNAs in miRBase 21,643 mature miRNAs - 168 plant, animal, and virus species miRBase: microRNA sequences, targets and gene nomenclature. Griffiths-Jones S et al. NAR 2006 34(Database Issue): D140-D144 [ article ]

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miRNAs in PubMed Subscribe to PubMed RSS feed for “microRNA AND cancer”

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Latest from the Funding Agencies NIH To Fund Projects to ID miRNA Cancer Markers, Study Role of ncRNAs in HIV-Associated Disorders Last month NIH announced that it is seeking research grant applications for projects related to the identification of microRNAs and other non-coding RNAs as biomarkers in the early detection of cancer . The funding agency is specifically encouraging research projects to assess the utility of stable miRNAs and ncRNAs to predict progression to cancer, it said. “Building on both basic and biomarker research on microRNAs, this [research opportunity] will further promote research on all classes of ncRNAs and support the translation of stable miRNAs into cancer screening or diagnostic tests .”

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Why Study miRNAs in Cancer? miRNAs regulate molecular pathways in cancer by targeting both oncogenes and tumour suppressors . miRNAs have a role in cancer-stem-cell biology , angiogenesis, the epithelial– mesenchymal transition, metastasis , and drug resistance . miRNA dysregulation is a crucial part of tumour: formation , maintenance , and metastasis . miRNAs play an essential part in malignancy , functioning as tumour suppressors and oncogenes . miR-21 is an example of an oncogenic miRNA ( oncomir ). It is overexpressed in most cancers—breast cancer, colorectal cancer, lung cancer, pancreatic cancer, glioblastoma , neuroblastoma , leukemia, and lymphoma. miR-15 and miR-16 are examples of tumour suppressors. They target the oncogene BCL2 , which encodes a protein involved in cell survival

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17 miRNAs in Cancer: Clinical Potential Molecular Diagnostics / Biomarkers – Identification of specific miRNAs or miRNA expression based signatures that can act as biomarkers for various diseases/pathologies. Biomarkers in body fluids Make accurate and detailed clinical diagnosis Determine prognosis and predict treatment efficacy Monitor and assess the carcinogenic effects of environmental and other toxicants Rosetta Genomics' miRview Lung Assay: Assay Shown to Accurately Differentiate Between the Four Main Types of Lung Cancer

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18 miRNAs in Cancer: Clinical Potential Drug Discovery / Therapeutics – Identification of miRNAs that play essential roles in disease to act as drugs or possible therapeutic targets inhibitors. miRNAs as tumor suppressor drugs miRNAs as drug targets Study of miRNAs to understand chemo/radio resistance

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19 Reviews: miRNAs in all types of Cancers MicroRNA and its roles in esophageal cancer . Fang Y et al. Med Sci Monit . 2012 Mar;18(3):RA22-30. [ abstract ] MicroRNA in HCV infection and liver cancer . Kumar A. Biochim Biophys Acta . 2011 Nov-Dec;1809(11-12):694-9. [ abstract ] MicroRNA and breast cancer -related hormone receptors. Zhang J et al. 2011 Mar;40(3):212-4. [ abstract ] MicroRNA in lung cancer diagnostics and treatment. Skrzypski M et al. Mutat Res. 2011 Dec 1;717(1-2):25-31. [ abstract ] MicroRNA dysregulation in colorectal cancer . Dong Y et al. Br J Cancer. 2011 Mar 15;104(6):893-8. [ abstract ] MicroRNA in prostate, bladder, and kidney cancer . Catto JW et al. Eur Urol. 2011 May;59(5):671-81. [ abstract ] MicroRNA dysregulation in gastric cancer . Wu WK et al. Oncogene . 2010 Oct 28;29(43):5761-71. [ abstract ] MicroRNA in pancreatic cancer . Rachagani S et al. Cancer Lett . 2010 Jun 1;292(1):8-16. [ abstract ] MicroRNA and oral cancer . Gomes CC et al. Oral Oncol . 2008 Oct;44(10):910-4. [ abstract ] MicroRNA and ovarian cancer . Corney DC et al. Histol Histopathol . 2008 Sep;23(9):1161-9. [ abstract ]

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20 Kong YW, Ferland-McCollough D, Jackson TJ, Bushell M. (2012) microRNAs in cancer management. Lancet Oncol 13(6):e249-58. [ abstract ] 2012 Review: miRNAs in Cancer Management This Review summarizes the present understanding of how miRNAs operate at the molecular level; how their dysregulation is a crucial part of tumor formation, maintenance, and metastasis; how they can be used as biomarkers for disease type and grade; and how miRNA-based treatments could be used for diverse types of malignancies. MicroRNAs are now known to play an essential part in malignancy, functioning as tumor suppressors and oncogenes

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MicroRNA profiling: approaches and considerations Colin C. Pritchard, Heather H. Cheng & Muneesh Tewari Nature Reviews Genetics 13, 358-369 (May 2012 ) .[ abstract ] MicroRNA Profiling Review:

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Seq-Array SM ACGT101-miR Stem-Loop Specific 22 Target Determination Detection & Profiling Functional Analysis Northern Blotting In situ hybridization Real-time PCR Microarray analysis Next-gen Sequencing Bioinformatics – TargetScan , PicTar , PITA Gene / proteome expression analysis Pull-down assays 5′ RACE analyses Degradome Sequencing Lucifierase Assays Gene knockout/overexpression models miRNA inhibition - antagomirs miRNA mimicry Degradsome Seq Digital Gene Expression Pathway Network miRNA Identification Genetic screening Direct cloning, sequencing Computational strategy – MIRCheck , findMiRNA , MIRscan , MiRAlign Tiling Microarrays Next-gen Sequencing Pathway Analysis Bioinformatics – miRFocus , mirPath , MMIA microRNA Research Tools

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miRNA microarray qRT -PCR custom miRNA microarray Next Gen Sequencing Discovery Profiling Quantitation Validation 3 Major Steps & Technologies } Seq-Array SM

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24 Sample Preparation Cell Line Tissue Blood Serum Plasma FFPE Block Algea Plant Material Fatty Tissue (Viscous Samples) Total RNA (1-4 µg) Norgen Biotek Ambion Qiagen S elect a kit designed to retain small RNA Select kit based on your sample type Use the same kit for entire experiment miRVana Kit miRNeasy Kit Total RNA Extraction Kit Urine

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You can check the UV spectrum of your sample with a spectrophotometer. 25 ↑ 1.0 ↑ 1.8 260nm 230nm 260nm 280nm Bioanalyzer or 1-1.5% agarose gel 28S rRNA band at 4.5kb - ~2X intensity 18S rRNA band at 1.9kb. For Average Cell Line or Tissue sample – RIN number must be ↑ 7 For other sample types such as Blood or Plant – RIN number does not apply Excessive smearing on the gel indicates degraded RNA. Customer Sample Quality Control

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26 Customer Sample Quality Control microRNA Microarray Expression Profiling Good Poor Good Poor Good Poor Failure in recovery of RNA <200 nt (including microRNA) 1.5% Formaldehyde Agarose Gel Agilent BioAnalyzer Gel Image Urea-PAGE Gel

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27 Biological Replicates – Still Very Important For experiments performed with a small number of biological replicates, significant results may be due to biological diversity between individuals and may not be reproducible - it is impossible to know whether expression patterns are specific to the individuals in the study or are a characteristic of the test condition. There is no statistical significance for a difference observed between 2 samples. There is no magic to RNA-Seq. These ideas are widely accepted for DNA microarray experiments, where a large number of biological replicates are now required to justify scientific conclusions . Hansen KD, Wu Z, Irizarry RA, Leek JT.  2011 Sequencing technology does not eliminate biological variability. Nat Biotechnol 29:572–573. [ abstract ] Reg. Experimental Design Sample Replicates for Expression Studies

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Key Advantages of RNA-Seq Provides a comprehensive view of the transcriptome. All transcripts can be analyzed (mRNA, ncRNA, snoRNA, lncRNA , miRNA, ...). Not necessarily dependent on any prior sequence knowledge. Increased dynamic range and tunable sensitivity . Can detect structural variations such as gene fusions and alternative splicing events. A truly digital solution (absolute abundance vs relative abundance). Microarray vs RNA Sequencing 28 Key Advantages of Microarray Robust, reliable method, proven over decades of use High through-put method – 100s of samples analyzed per month Streamlined handling – can be easily automated Straightforward data analysis Short turn-around time – 5 days Lower cost

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Microfluidic Array Platform μ Paraflo ® Microfluidics Chip 10 µl total volume 4000 features 270 pl / reaction chamber uniform flow rate

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miRBase Version # of precursor miRNA sequences (all species) Flexibility Allows miRBase Synchronicity Version 18 Nov 2011 2003 2011

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31 Comprehensive Microarray Services Sample QC Sample preparation Hybridization reactions Advanced data analysis High level customer support Customer Total RNA Small RNA Isolation Labeling Customer Sequences Chip Design Chip Synthesis Chip QC Hybridization Signal Amplification Image Acquisition Customer Analysis Report Data Extraction Data Analysis Sample QC miRBase microRNA Microarray Expression Profiling

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Control / Treated Biological repeats t-Test p < 0.05 p < 0.01 Control Treated Multi-array normalization and clustering analysis Array assay Differentiated miRNAs of Biological & Statistical Significance - Multiple Chips 32 microRNA Microarray Expression Profiling

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Repeats microRNA Microarray Expression Profiling

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34 Advanced Data Analysis Package Includes: The original and processed chip images An array layout file A raw intensity data file in Excel A fully analyzed data file in Excel A Data Summary containing a catalog of data files, images Images of representative regions of corresponding arrays Descriptions of specific features of the arrays A list of up and down regulated transcripts that are called based on a statistical analysis. microRNA Microarray Expression Profiling

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Key Advantages of RNA-Seq Provides a comprehensive view of the transcriptome. All transcripts can be analyzed (mRNA, ncRNA, snoRNA, lncRNA , miRNA, ...). Not necessarily dependent on any prior sequence knowledge. Increased dynamic range and tunable sensitivity . Can detect structural variations such as gene fusions and alternative splicing events. A truly digital solution (absolute abundance vs relative abundance). Microarray vs RNA Sequencing 35 Key Advantages of Microarray Robust, reliable method, proven over decades of use High through-put method – 100s of samples analyzed per month Streamlined handling – can be easily automated Straightforward data analysis Short turn-around time – 5 days Lower cost

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36 Small RNA Sequencing and Data Analysis LC Sciences - Comprehensive RNA Sequencing Services Sample QC Sample preparation Library preparation High-throughput sequencing Advanced bioinformatics analysis High level customer support

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37 Sequencing Run Instrument: Illumina Genome Analyzer GAIIx Length of Reads: 35 bases Number of Reads: ~20-30 Million Data Output: ~0.7-1.0 Gb Bar-coding (Indexing) Samples: We recommend 3 per lane, Max is 6 per lane The total number of reads does not change with bar-coding Sacrifice sequencing depth for lower cost Total Reads / Lane Number of Samples / Lane Reads/ Sample 30 M 1 30 M 30 M 2 15 M 30 M 3 10 M 30 M 4 7.5 M 30 M 5 6 M 30 M 6 5 M Small RNA Sequencing and Data Analysis

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Data Flow 38 Mappable reads Raw reads Reads mapped to mammalian mirs in miRBase Reads un-mapped to mammalian mirs in miRBase mirs mapped to species genome mirs un-mapped to species genome Reads mapped to species genome Reads un-mapped to species genome Reads un-mapped to mRNA, Rfam, and repbase Reads mapped to mRNA, Rfam, and repbase Reads mapped to species genome Reads un-mapped to species genome Group 4 no hit others Group 1 Group 2 Group 3 Known species miRNAs Known miRNAs candidate species miRNAs Candidate species miRNAs genome inconsistent with miRBase Potentially novel miRNAs ACGT101-miR v3.5 Software Small RNA Sequencing and Data Analysis

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39 19,842,938 reads are mappable ADT, Junk, & Seq Filter 11,426,638 reads are mapped to miRBase or are miRNA candidates 2,400,521 reads mapped to mRNA, Rfam and repbase 6,015,779 reads - no hit 10,713,874 reads are filtered out 30,556,812 raw reads from sequencer Grp 1 - 8,007,998 Grp 2 - 14,067 Grp 3 - 64,086 Grp 4 - 3,340,487 64.9% 19.7% 15.1% 0.3% 57.6% 30.3% 12.1% 70.1% 29.2% 0.1% 0.6% Data Flow Small RNA Sequencing and Data Analysis

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40 Length Distribution of Mappable Reads Small RNA Sequencing and Data Analysis

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Can explain discrepancies array data vs qRT-PCR validation 41 IsomiRs Small RNA Sequencing and Data Analysis

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Molecular Diagnostics / Biomarkers in Body Fluids – Plasma miRNA profiling of plasma may provide a molecular signature for tumor aggressiveness in pancreatic cancer Ali S, Almhanna K, Chen W, Philip PA, Sarkar FH. (2010) Differentially expressed miRNAs in the plasma may provide a molecular signature for aggressive pancreatic cancer . Am J Transl Res 3(1), 28-47 . [ article ] Pancreatic cancer ( PC): Comparison of microRNA expression profiles in the plasma of patients diagnosed with PC (n=50) with healthy volunteers (n=10). Data was further validated by qTR -PCR and cell-based assays. 37 miRNAs down-regulated 54 miRNAs up-regulated Expression of miR-21 was correlated with worse survival Expression of let-7 was inversely correlated with survival miR-21 family was markedly over-expressed in chemo-resistant PC cell lines

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Release of miRNAs from cells into breast milk is selective and that the selection of released miRNAs may correlate with malignancy Pigati L, Yaddanapudi SC, Iyengar R, Kim DJ, Hearn SA, Danforth D, Hastings ML, Duelli DM. (2010) Selective release of microRNA species from normal and malignant mammary epithelial cells. PLoS ONE . 5(10), e13515 . [ article ] Compared microRNA of MCF7 cellular and extracellular RNAs via microarray analysis Total RNA was isolated from exosomes of MCF7 cells or milk of healthy mothers. The bulk of miR-451 and miR-1246 produced by malignant mammary epithelial cells was released, but the majority of these miRNAs produced by non-malignant mammary epithelial cells was retained. Milk contains extracellular miRNA species originating from mammary epithelial cells in a specific conserved ratio and that the selection of released miRNAs may correlate with malignancy. Duplicate microRNA microarrays were hybridized with 1 µg of total cellular or 1 µg of extracellular miRNA from MCF7 cells. Results are plotted as A relative fluorescent intensities of extracellular (x, upper panel) and cellular (c, lower panel) miRNAs, or B ratio of extracellular to cellular miRNAs. C Scatter plot of average reads of the miRNAs quantitated by array. Molecular Diagnostics / Biomarkers in Body Fluids – Breast Milk

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Biomarkers – Molecular Diagnostics – Diagnosis miRNA expression patterns can distinguish between renal cell carcinoma subtypes YM Youssef , N White, J Grigull , A Krizova , C Samy et al. (2011) Accurate Molecular Classification of Kidney Cancer Subtypes Using MicroRNA Signature . European Urology 59(5), 721-30. [ article ] Kidney Cancer: Renal cell carcinoma (RCC) encompasses different histologic subtypes. Distinguishing between the subtypes is usually made by morphologic assessment, which is not always accurate. A total of 94 different subtype cases were analyzed. miRNA microarray analysis was performed on fresh frozen tissues of three common RCC subtypes (clear cell, chromophobe, and papillary) and on oncocytoma. Results were validated on the original as well as on an independent set of tumours, using qRT-PCR analysis with miRNA specific primers. Fig 1 - A hierarchic cluster heat map showing differential miRNA expression in normal kidney, oncocytoma, and different RCC subtypes.

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Biomarkers – Molecular Diagnostics - Prognosis miR-150 as a potential biomarker associated with prognosis and therapeutic outcome in colorectal cancer Ma Y, Zhang P, Wang F, Zhang H, Yang J, Peng J, Liu W, Qin H. (2011) miR-150 as a potential biomarker associated with prognosis and therapeutic outcome in colorectal cancer . Gut [Epub ahead of print]. [ abstract ] Colorectal C ancer (CRC) Up to 90% of patients with colorectal cancer (CRC), one of the most common malignancies in the Western world , can be cured by surgery if the disease is detected at an early stage. However, it is often diagnosed only at an advanced stage and the prognosis is therefore poor. Performed global analysis of miRNA expression profiles of normal colorectal tissues, colorectal adenoma tissues and CRC tissues miRNA microarray. Study then focused on only one miRNA ( miR-150 ) because its expression decreased with the transition from normal mucosa via adenoma to CRC in parallel with increasing carcinogenesis of the colorectal tissue. Found that the miR-150 expression status of patients with CRC is associated with survival and response to adjuvant chemotherapy is therefore a potential biomarker associated with the prognosis and therapeutic outcome in CRC.

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Biomarkers – Molecular Diagnostics Exposure to Toxicants miRNA expression profiles distinguish the carcinogenic effects of riddelliine in rat liver Chen T, Li Z, Yan J, Yang X, Salminen W. (2011) MicroRNA expression profiles distinguish the carcinogenic effects of riddelliine in rat liver . Mutagenesis 27(1), 59-66. [ abstract ] Pyrrolizidine alkaloids (PAs) are the most common plant constituents that poison livestock, wildlife and humans. Riddelliine , a genotoxic PA, has been nominated to be classified as a reasonably anticipated human carcinogen by the USNTP. miRNA expression profiling with miRNA microarrays and subsequent principal component analysis and hierarchical clustering analysis showed that the miRNA expression profiles were clearly classified into two groups, riddelliine treatment versus other samples . 47 miRNAs were significantly dysregulated by riddelliine treatment . Potential use of miRNAs as biomarker of genotoxicity and carcinogenicity for riddelliine and possibly other Pas. CONTROL RIDDELLINE

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Drug Discovery / Therapeutics miRNAs as Tumor Suppressors Microarray based miRNA profiling of highly metastatic human breast cancer cell derivatives has yielded a set of microRNAs for which expression is specifically lost as human breast cancer cells develop metastatic potential. Show that restoring the expression of these microRNAs in malignant cells suppresses lung and bone metastasis by human cancer cells in vivo . miR-126 restoration reduces overall tumour growth and proliferation, whereas miR-335 inhibits metastatic cell invasion. Expression of miR-126 and miR-335 is lost in the majority of primary breast tumours from patients who relapse, and the loss of expression of either microRNA is associated with poor distal metastasis-free survival. miR-335 and miR-126 are thus identified as metastasis suppressor microRNAs in human breast cancer. Tavazoie SF, Alarcón C, Oskarsson T, Padua D, Wang Q, Bos PD, Gerald WL, Massagué J. (2008) Endogenous human microRNAs that suppress breast cancer metastasis . Nature 451(7175), 147-52 . [ abstract ]

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Drug Discovery / Therapeutics Chemotherapy Resistance Restoring miR-342 expression may represent a novel therapeutic approach to sensitizing and suppressing the growth of tamoxifen resistant breast tumors Cittelly DM, Das PM, Spoelstra NS, Edgerton SM, Richer JK, Thor AD, Jones FE. (2010) Downregulation of miR-342 is Associated with Tamoxifen Resistant Breast Tumors. Mol Cancer 9(1), 317. [ article ] Tumor resistance to the selective estrogen receptor modulator tamoxifen remains a serious clinical problem especially in patients with tumors that also overexpress HER2. However, a unifying molecular mechanism of tamoxifen resistance has remained elusive. Analyzed multiple cell models of tamoxifen resistance derived from MCF-7 cells to examine the influence of miRNAs on tamoxifen resistance. Observed significant and dramatic downregulation of miR-342 in tamoxifen resistant MCF-7 variant cell lines. Restoring miR-342 expression in the cell lines sensitized these cells to tamoxifen -induced apoptosis with a dramatic reduction in cell growth.

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