An estimate of the total number of true human miRNAs

Abstract While the number of human miRNA candidates continuously increases, only a few of them are completely characterized and experimentally validated. Toward determining the total number of true miRNAs, we employed a combined in silico high- and experimental low-throughput validation strategy. We collected 28 866 human small RNA sequencing data sets containing 363.7 billion sequencing reads and excluded falsely annotated and low quality data. Our high-throughput analysis identified 65% of 24 127 mature miRNA candidates as likely false-positives. Using northern blotting, we experimentally validated miRBase entries and novel miRNA candidates. By exogenous overexpression of 108 precursors that encode 205 mature miRNAs, we confirmed 68.5% of the miRBase entries with the confirmation rate going up to 94.4% for the high-confidence entries and 18.3% of the novel miRNA candidates. Analyzing endogenous miRNAs, we verified the expression of 8 miRNAs in 12 different human cell lines. In total, we extrapolated 2300 true human mature miRNAs, 1115 of which are currently annotated in miRBase V22. The experimentally validated miRNAs will contribute to revising targetomes hypothesized by utilizing falsely annotated miRNAs.

[1]  T. Smart,et al.  HEK293 cell line: a vehicle for the expression of recombinant proteins. , 2005, Journal of pharmacological and toxicological methods.

[2]  D. Galetta,et al.  miRNA profiling in serum and tissue samples to assess noninvasive biomarkers for NSCLC clinical outcome , 2016, Tumor Biology.

[3]  D. Xie,et al.  Decreased expression of miR-939 contributes to chemoresistance and metastasis of gastric cancer via dysregulation of SLC34A2 and Raf/MEK/ERK pathway , 2017, Molecular Cancer.

[4]  A. Meyerhans,et al.  DNA recombination during PCR. , 1990, Nucleic acids research.

[5]  Hongbing Shen,et al.  Plasma miRNAs as early biomarkers for detecting hepatocellular carcinoma , 2015, International journal of cancer.

[6]  D. Bartel.  MicroRNAs: Target Recognition and Regulatory Functions , 2009, Cell.

[7]  Y. Liu,et al.  MicroRNA-939 governs vascular integrity and angiogenesis through targeting γ-catenin in endothelial cells. , 2017, Biochemical and biophysical research communications.

[8]  Christina Backes,et al.  A high‐resolution map of the human small non‐coding transcriptome , 2018, Bioinform..

[9]  C. Nusbaum,et al.  Mammalian microRNAs: experimental evaluation of novel and previously annotated genes. , 2010, Genes & development.

[10]  Thomas Tuschl,et al.  Mammalian miRNA curation through next-generation sequencing , 2013, Front. Genet..

[11]  G. Meister,et al.  miRNA expression profiling of Epstein–Barr virus‐associated NKTL cell lines by Illumina deep sequencing , 2016, FEBS open bio.

[12]  Eckart Meese,et al.  Can circulating miRNAs live up to the promise of being minimal invasive biomarkers in clinical settings? , 2016, Wiley interdisciplinary reviews. RNA.

[13]  R. Jove,et al.  A Novel Berbamine Derivative Inhibits Cell Viability and Induces Apoptosis in Cancer Stem-Like Cells of Human Glioblastoma, via Up-Regulation of miRNA-4284 and JNK/AP-1 Signaling , 2014, PloS one.

[14]  J. Kjems,et al.  Argonaute-associated short introns are a novel class of gene regulators , 2016, Nature Communications.

[15]  M. Behlke,et al.  Modified Polyadenylation-Based RT-qPCR Increases Selectivity of Amplification of 3′-MicroRNA Isoforms , 2018, Front. Genet..

[16]  Ana Kozomara,et al.  miRBase: integrating microRNA annotation and deep-sequencing data , 2010, Nucleic Acids Res..

[17]  U. Fischer,et al.  Panel of five microRNAs as potential biomarkers for the diagnosis and assessment of male infertility. , 2014, Fertility and sterility.

[18]  Sam Griffiths-Jones,et al.  The microRNA Registry , 2004, Nucleic Acids Res..

[19]  A. Hartmann,et al.  Comparative microRNA Profiling of Prostate Carcinomas with Increasing Tumor Stage by Deep Sequencing , 2013, Molecular Cancer Research.

[20]  P. Khaitovich,et al.  Sequence features associated with microRNA strand selection in humans and flies , 2009, BMC Genomics.

[21]  X. Shirley Liu,et al.  Systematic Curation of miRBase Annotation Using Integrated Small RNA High-Throughput Sequencing Data for C. elegans and Drosophila , 2011, Front. Gene..

[22]  Stefano Volinia,et al.  MicroRNA Profiles Discriminate among Colon Cancer Metastasis , 2014, PloS one.

[23]  A. Sureau,et al.  Short intron-derived ncRNAs , 2017, Nucleic acids research.

[24]  J. Reiss,et al.  Polymerase chain reaction: replication errors and reliability of gene diagnosis. , 1989, Nucleic acids research.

[25]  Aristeidis G. Telonis,et al.  Assessment of isomiR Discrimination Using Commercial qPCR Methods , 2017, Non-coding RNA.

[26]  J. Haas,et al.  Prioritizing and selecting likely novel miRNAs from NGS data , 2015, Nucleic Acids Research.

[27]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[28]  Ana Kozomara,et al.  miRBase: annotating high confidence microRNAs using deep sequencing data , 2013, Nucleic Acids Res..

[29]  D. Berg,et al.  Validating Alzheimer's disease micro RNAs using next-generation sequencing , 2016, Alzheimer's & Dementia.

[30]  A. Keller,et al.  Distribution of miRNA expression across human tissues , 2016, Nucleic acids research.

[31]  Sabine C. Mueller,et al.  A blood based 12-miRNA signature of Alzheimer disease patients , 2013, Genome Biology.

[32]  Ming Chen,et al.  Are all the miRBase-registered microRNAs true? , 2012, RNA biology.

[33]  V. Ambros.  The functions of animal microRNAs , 2004, Nature.

[34]  F. Graham,et al.  Characteristics of a human cell line transformed by DNA from human adenovirus type 5. , 1977, The Journal of general virology.

[35]  M. Soleimani,et al.  Overexpression of hsa-miR-939 follows by NGFR down-regulation and apoptosis reduction , 2017, Journal of Biosciences.

[36]  Christina Backes,et al.  Toward the blood-borne miRNome of human diseases , 2011, Nature Methods.

[37]  S. Bortoluzzi,et al.  Impact of Host Genes and Strand Selection on miRNA and miRNA* Expression , 2011, PloS one.

[38]  D. Galasko,et al.  A cerebrospinal fluid microRNA signature as biomarker for glioblastoma , 2017, Oncotarget.

[39]  Ana Kozomara,et al.  miRBase: from microRNA sequences to function , 2018, Nucleic Acids Res..

[40]  Hans-Peter Lenhof,et al.  Circulating serum miRNAs as potential biomarkers for nephroblastoma , 2015, Pediatric blood & cancer.

[41]  Christina Backes,et al.  miRCarta: a central repository for collecting miRNA candidates , 2017, Nucleic Acids Res..

[42]  B. Sarkadi,et al.  Human mirtrons can express functional microRNAs simultaneously from both arms in a flanking exon-independent manner , 2012, RNA biology.

[43]  Analysis of circulating microRNAs in patients with repaired Tetralogy of Fallot with and without heart failure , 2017, Journal of Translational Medicine.

[44]  S. Jayasena,et al.  Functional siRNAs and miRNAs Exhibit Strand Bias , 2003, Cell.

[45]  V. Kim,et al.  Re-evaluation of the roles of DROSHA, Exportin 5, and DICER in microRNA biogenesis , 2016, Proceedings of the National Academy of Sciences.

[46]  MicroRNA-4284 promotes gastric cancer tumorigenicity by targeting ten-eleven translocation 1 , 2018, Molecular medicine reports.

[47]  C. Bracken,et al.  miRNA length variation during macrophage stimulation confounds the interpretation of results: implications for miRNA quantification by RT-qPCR , 2018, RNA.

[48]  Min Zhang,et al.  MicroRNA-939 restricts Hepatitis B virus by targeting Jmjd3-mediated and C/EBPα-coordinated chromatin remodeling , 2016, Scientific Reports.

[49]  Sebastian D. Mackowiak,et al.  miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades , 2011, Nucleic acids research.

[50]  Andreas E. Posch,et al.  Web-based NGS data analysis using miRMaster: a large-scale meta-analysis of human miRNAs , 2017, Nucleic acids research.

[51]  E. Hovig,et al.  A Uniform System for the Annotation of Vertebrate microRNA Genes and the Evolution of the Human microRNAome. , 2015, Annual review of genetics.

[52]  Bino John,et al.  miRNA-939 regulates human inducible nitric oxide synthase posttranscriptional gene expression in human hepatocytes , 2012, Proceedings of the National Academy of Sciences.

[53]  Rasko Leinonen,et al.  The sequence read archive: explosive growth of sequencing data , 2011, Nucleic Acids Res..

[54]  J. Kjems,et al.  Enhancing miRNA annotation confidence in miRBase by continuous cross dataset analysis , 2011, RNA biology.