Circular RNA expression is abundant and correlated to aggressiveness in early-stage bladder cancer

The functions and biomarker potential of circular RNAs (circRNAs) in various cancer types are a rising field of study, as emerging evidence relates circRNAs to tumorigenesis. Here, we profiled the expression of circRNAs in 457 tumors from patients with non-muscle-invasive bladder cancer (NMIBC). We show that a set of highly expressed circRNAs have conserved core splice sites, are associated with Alu repeats, and enriched with Synonymous Constraint Elements as well as microRNA target sites. We identified 113 abundant circRNAs that are differentially expressed between high and low-risk tumor subtypes. Analysis of progression-free survival revealed 13 circRNAs, among them circHIPK3 and circCDYL, where expression correlated with progression independently of the linear transcript and the host gene. In summary, our results demonstrate that abundant circRNAs possess multiple biological features, distinguishing them from low-expressed circRNAs and non-circularized exons, and suggest that circRNAs might serve as a new class of prognostic biomarkers in NMIBC.Bladder cancer: Circular RNAs show prognostic biomarker potentialExpression levels of non-coding “circular” RNA molecules could be used as a prognostic biomarker for patients with early-stage bladder cancer. A team led by Trine Line Hauge Okholm and Jakob Skou Pedersen from Aarhus University Hospital, Denmark, profiled the expression of these loop-forming, potentially gene-regulating RNAs in biopsied tumor samples from 457 patients with bladder cancer that had not invaded nearby muscle tissue. They identified a suite of 113 circular RNAs that were abundant and differentially expressed between patients with different molecular subtypes of bladder cancer. The researchers also found a smaller set of 13 circular RNAs for which expression levels correlated with disease progression. These non-coding RNA molecules, by indicating likely patient outcomes, could potentially serve as future diagnostic aids to inform treatment strategies and decisions.

[1]  Fengling Wang,et al.  Circular RNAs as potential biomarkers for cancer diagnosis and therapy. , 2016, American journal of cancer research.

[2]  Ming Yi,et al.  miRNA-7 attenuation in Schwannoma tumors stimulates growth by upregulating three oncogenic signaling pathways. , 2011, Cancer research.

[3]  D. Bartel,et al.  Expanded identification and characterization of mammalian circular RNAs , 2014, Genome Biology.

[4]  Tim Schneider,et al.  Exon circularization requires canonical splice signals. , 2015, Cell reports.

[5]  A. Dreher.  Modeling Survival Data Extending The Cox Model , 2016 .

[6]  Manolis Kellis,et al.  Locating protein-coding sequences under selection for additional, overlapping functions in 29 mammalian genomes. , 2011, Genome research.

[7]  Ling Tian,et al.  MicroRNA‐7 inhibits tumor growth and metastasis by targeting the phosphoinositide 3‐kinase/Akt pathway in hepatocellular carcinoma , 2012, Hepatology.

[8]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[9]  N. Rajewsky,et al.  Translation of CircRNAs , 2017, Molecular cell.

[10]  Andrew D. Smith,et al.  Site identification in high-throughput RNA-protein interaction data , 2012, Bioinform..

[11]  J Alfred Witjes,et al.  Predicting recurrence and progression in individual patients with stage Ta T1 bladder cancer using EORTC risk tables: a combined analysis of 2596 patients from seven EORTC trials. , 2006, European urology.

[12]  Alex E. Lash,et al.  Gene Expression Omnibus: NCBI gene expression and hybridization array data repository , 2002, Nucleic Acids Res..

[13]  N. Sharpless,et al.  Detecting and characterizing circular RNAs , 2014, Nature Biotechnology.

[14]  Toshiyoshi Fujiwara,et al.  Circular RNA ciRS-7—A Promising Prognostic Biomarker and a Potential Therapeutic Target in Colorectal Cancer , 2017, Clinical Cancer Research.

[15]  Michael K. Slevin,et al.  Circular RNAs are abundant, conserved, and associated with ALU repeats. , 2013, RNA.

[16]  Paul Theodor Pyl,et al.  HTSeq—a Python framework to work with high-throughput sequencing data , 2014, bioRxiv.

[17]  Shinji Tanaka,et al.  miR-124 and miR-203 are epigenetically silenced tumor-suppressive microRNAs in hepatocellular carcinoma. , 2010, Carcinogenesis.

[18]  J. Kjems,et al.  Circular RNA and miR-7 in cancer. , 2013, Cancer research.

[19]  C. Cole,et al.  COSMIC: High‐Resolution Cancer Genetics Using the Catalogue of Somatic Mutations in Cancer , 2016, Current protocols in human genetics.

[20]  M. Babjuk,et al.  EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder: update 2013. , 2013, European urology.

[21]  C. Burge,et al.  Conserved Seed Pairing, Often Flanked by Adenosines, Indicates that Thousands of Human Genes are MicroRNA Targets , 2005, Cell.

[22]  Fuqing Zeng,et al.  CircHIPK3 sponges miR‐558 to suppress heparanase expression in bladder cancer cells , 2017, EMBO reports.

[23]  Cole Trapnell,et al.  Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. , 2010, Nature biotechnology.

[24]  Ping Yang,et al.  Silencing of cZNF292 circular RNA suppresses human glioma tube formation via the Wnt/β-catenin signaling pathway , 2016, Oncotarget.

[25]  Bin Li,et al.  Circular RNAs in cancer: an emerging key player , 2017, Journal of Hematology & Oncology.

[26]  E. Schuman,et al.  Neural circular RNAs are derived from synaptic genes and regulated by development and plasticity , 2015, Nature Neuroscience.

[27]  N. Rajewsky,et al.  circRNA biogenesis competes with pre-mRNA splicing. , 2014, Molecular cell.

[28]  Feipeng Zhao,et al.  MiR-124 suppresses tumor growth and metastasis by targeting Foxq1 in nasopharyngeal carcinoma , 2014, Molecular Cancer.

[29]  P. Grambsch,et al.  A Package for Survival Analysis in S , 1994 .

[30]  E. Larsson,et al.  Systematic analysis of noncoding somatic mutations and gene expression alterations across 14 tumor types , 2014, Nature Genetics.

[31]  Sebastian D. Mackowiak,et al.  Circular RNAs are a large class of animal RNAs with regulatory potency , 2013, Nature.

[32]  Julia Salzman,et al.  Cell-Type Specific Features of Circular RNA Expression , 2013, PLoS genetics.

[33]  Tom H. Pringle,et al.  The human genome browser at UCSC. , 2002, Genome research.

[34]  J. Stone,et al.  Radical Cystectomy for Invasive Bladder Cancer , 1966 .

[35]  D. Wheeler,et al.  Identification of a pan-cancer oncogenic microRNA superfamily anchored by a central core seed motif , 2013, Nature Communications.

[36]  C. Luo,et al.  Differential Expression of Circular RNAs in Glioblastoma Multiforme and Its Correlation with Prognosis , 2017, Translational oncology.

[37]  P. Brown,et al.  Circular RNA Is Expressed across the Eukaryotic Tree of Life , 2014, PloS one.

[38]  C. Mathers,et al.  Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012 , 2015, International journal of cancer.

[39]  J. Kjems,et al.  Natural RNA circles function as efficient microRNA sponges , 2013, Nature.

[40]  Jun Wang,et al.  Comprehensive characterization of tissue-specific circular RNAs in the human and mouse genomes , 2016, Briefings Bioinform..

[41]  K. Ohshiro,et al.  MicroRNA-7, a homeobox D10 target, inhibits p21-activated kinase 1 and regulates its functions. , 2008, Cancer research.

[42]  Yan Li,et al.  Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs , 2016, Nature Communications.

[43]  Laising Yen,et al.  Noncoding Effects of Circular RNA CCDC66 Promote Colon Cancer Growth and Metastasis. , 2017, Cancer research.

[44]  Hecheng Zhou,et al.  CircRNA: functions and properties of a novel potential biomarker for cancer , 2017, Molecular Cancer.

[45]  Steven L Salzberg,et al.  Fast gapped-read alignment with Bowtie 2 , 2012, Nature Methods.

[46]  John S Mattick,et al.  Regulation of Epidermal Growth Factor Receptor Signaling in Human Cancer Cells by MicroRNA-7* , 2009, Journal of Biological Chemistry.

[47]  Weining Yang,et al.  Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2 , 2016, Nucleic acids research.

[48]  N. Rajewsky,et al.  Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis , 2017, Molecular cell.

[49]  M. L. Calle,et al.  Comprehensive Transcriptional Analysis of Early-Stage Urothelial Carcinoma. , 2016, Cancer cell.

[50]  Maximilian Burger,et al.  EAU Guidelines on Non-Muscle-invasive Urothelial Carcinoma of the Bladder: Update 2016. , 2017, European urology.

[51]  Junxia Chen,et al.  Screening differential circular RNA expression profiles reveals the regulatory role of circTCF25-miR-103a-3p/miR-107-CDK6 pathway in bladder carcinoma , 2016, Scientific Reports.

[52]  Tobias Madsen,et al.  Non-coding cancer driver candidates identified with a sample- and position-specific model of the somatic mutation rate , 2017, eLife.

[53]  Peter Dalgaard,et al.  R Development Core Team (2010): R: A language and environment for statistical computing , 2010 .

[54]  Nikolaus Rajewsky,et al.  Identification and Characterization of Circular RNAs As a New Class of Putative Biomarkers in Human Blood , 2015, PloS one.

[55]  C. Cocquerelle,et al.  Mis‐splicing yields circular RNA molecules , 1993, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[56]  J. Kjems,et al.  Spatio-temporal regulation of circular RNA expression during porcine embryonic brain development , 2015, Genome Biology.

[57]  Huajie Cai,et al.  cir-ITCH Plays an Inhibitory Role in Colorectal Cancer by Regulating the Wnt/β-Catenin Pathway , 2015, PloS one.

[58]  Cedric E. Ginestet.  ggplot2: Elegant Graphics for Data Analysis , 2011 .

[59]  Yunqing Li,et al.  microRNA-7 inhibits the epidermal growth factor receptor and the Akt pathway and is down-regulated in glioblastoma. , 2008, Cancer research.

[60]  Christoph Dieterich,et al.  Analysis of intron sequences reveals hallmarks of circular RNA biogenesis in animals. , 2015, Cell reports.

[61]  A. Smit.  Interspersed repeats and other mementos of transposable elements in mammalian genomes. , 1999, Current opinion in genetics & development.

[62]  Hong Chen,et al.  miRNA-124 modulates lung carcinoma cell migration and invasion. , 2016, International journal of clinical pharmacology and therapeutics.

[63]  Yun Chen,et al.  Circular BANP, an upregulated circular RNA that modulates cell proliferation in colorectal cancer. , 2017, Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie.

[64]  Charles Gawad,et al.  Circular RNAs Are the Predominant Transcript Isoform from Hundreds of Human Genes in Diverse Cell Types , 2012, PloS one.

[65]  K. Pollard,et al.  Detection of nonneutral substitution rates on mammalian phylogenies. , 2010, Genome research.

[66]  Roy Parker,et al.  Circular RNAs Co-Precipitate with Extracellular Vesicles: A Possible Mechanism for circRNA Clearance , 2016, PloS one.

[67]  Jiang-xia Zhao,et al.  Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis , 2015, Cell Research.

[68]  Martin Renqiang Min,et al.  An integrated encyclopedia of DNA elements in the human genome , 2012 .

[69]  Tuan Dinh,et al.  The Health Economics of Bladder Cancer: An Updated Review of the Published Literature , 2014, PharmacoEconomics (Auckland).

[70]  Cristian Coarfa,et al.  The Landscape of microRNA Targeting in Prostate Cancer Defined by AGO-PAR-CLIP1 , 2016, Neoplasia.

[71]  S. Horenblas,et al.  Landmarks in non-muscle-invasive bladder cancer , 2014, Nature Reviews Urology.

[72]  Andreas W. Schreiber,et al.  The RNA Binding Protein Quaking Regulates Formation of circRNAs , 2015, Cell.

[73]  Yifeng Zhou,et al.  Circular RNA ITCH has inhibitory effect on ESCC by suppressing the Wnt/β-catenin pathway , 2015, Oncotarget.

[74]  Yan Guo,et al.  Heatmap3: an improved heatmap package with more powerful and convenient features , 2014, BMC Bioinformatics.

[75]  Data production leads,et al.  An integrated encyclopedia of DNA elements in the human genome , 2012 .

[76]  Cole Trapnell,et al.  TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions , 2013, Genome Biology.

[77]  Sergio Ricci,et al.  Long-term survival results of a randomized trial comparing gemcitabine plus cisplatin, with methotrexate, vinblastine, doxorubicin, plus cisplatin in patients with bladder cancer. , 2006, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.