Toward the promise of microRNAs – Enhancing reproducibility and rigor in microRNA research

ABSTRACT The fields of applied and translational microRNA research have exploded in recent years as microRNAs have been implicated across a spectrum of diseases. MicroRNA biomarkers, microRNA therapeutics, microRNA regulation of cellular physiology and even xenomiRs have stimulated great interest, which have brought many researchers into the field. Despite many successes in determining general mechanisms of microRNA generation and function, the application of microRNAs in translational areas has not had as much success. It has been a challenge to localize microRNAs to a given cell type within tissues and assay them reliably. At supraphysiologic levels, microRNAs may regulate hosts of genes that are not the physiologic biochemical targets. Thus the applied and translational microRNA literature is filled with pitfalls and claims that are neither scientifically rigorous nor reproducible. This review is focused on increasing awareness of the challenges of working with microRNAs in translational research and recommends better practices in this area of discovery.

[1]  K. Otsu,et al.  MicroRNA-451 Exacerbates Lipotoxicity in Cardiac Myocytes and High-Fat Diet-Induced Cardiac Hypertrophy in Mice Through Suppression of the LKB1/AMPK Pathway , 2015, Circulation research.

[2]  Kavitha T. Kuppusamy,et al.  Let-7 family of microRNA is required for maturation and adult-like metabolism in stem cell-derived cardiomyocytes , 2015, Proceedings of the National Academy of Sciences.

[3]  Bo W. Han,et al.  Circulating microRNAs identified in a genome-wide serum microRNA expression analysis as noninvasive biomarkers for endometriosis. , 2013, The Journal of clinical endocrinology and metabolism.

[4]  D. Tollervey,et al.  Mapping the Human miRNA Interactome by CLASH Reveals Frequent Noncanonical Binding , 2013, Cell.

[5]  K. Witwer,et al.  Real-time quantitative PCR and droplet digital PCR for plant miRNAs in mammalian blood provide little evidence for general uptake of dietary miRNAs Limited evidence for general uptake of dietary plant xenomiRs , 2013 .

[6]  T. Janas,et al.  Mechanisms of RNA loading into exosomes , 2015, FEBS letters.

[7]  C. Mantri,et al.  Cocaine Enhances HIV-1 Replication in CD4+ T Cells by Down-Regulating MiR-125b , 2012, PloS one.

[8]  E. van Rooij,et al.  Transient but Not Genetic Loss of miR-451 is Protective in the Development of Pulmonary Arterial Hypertension , 2013, Pulmonary circulation.

[9]  Xinchao Zhang,et al.  The downregulation of miR-144 is associated with the growth and invasion of osteosarcoma cells through the regulation of TAGLN expression. , 2014, International journal of molecular medicine.

[10]  Receptive to replication , 2013, Nature Biotechnology.

[11]  E. Kroh,et al.  Plasma Processing Conditions Substantially Influence Circulating microRNA Biomarker Levels , 2013, PloS one.

[12]  Anton J. Enright,et al.  Human MicroRNA Targets , 2004, PLoS biology.

[13]  Huiming Yu,et al.  miR‐144‐3p exerts anti‐tumor effects in glioblastoma by targeting c‐Met , 2015, Journal of neurochemistry.

[14]  J. Zempleni,et al.  MicroRNAs are absorbed in biologically meaningful amounts from nutritionally relevant doses of cow milk and affect gene expression in peripheral blood mononuclear cells, HEK-293 kidney cell cultures, and mouse livers. , 2014, The Journal of nutrition.

[15]  C. Thompson,et al.  Long-lived microRNA–Argonaute complexes in quiescent cells can be activated to regulate mitogenic responses , 2012, Proceedings of the National Academy of Sciences.

[16]  L. Donehower,et al.  Cross-species identification of a plasma microRNA signature for detection, therapeutic monitoring, and prognosis in osteosarcoma , 2015, Cancer medicine.

[17]  F. Liu,et al.  VEGF-activated miR-144 regulates autophagic survival of prostate cancer cells against Cisplatin , 2016, Tumor Biology.

[18]  Joana A. Vidigal,et al.  In vivo, Argonaute-bound microRNAs exist predominantly in a reservoir of low molecular weight complexes not associated with mRNA , 2015, Proceedings of the National Academy of Sciences.

[19]  K. Witwer Contamination or artifacts may explain reports of plant miRNAs in humans. , 2015, The Journal of nutritional biochemistry.

[20]  Athanasios Fevgas,et al.  DIANA-TarBase v7.0: indexing more than half a million experimentally supported miRNA:mRNA interactions , 2014, Nucleic Acids Res..

[21]  X. Chen,et al.  Effective detection and quantification of dietetically absorbed plant microRNAs in human plasma. , 2015, The Journal of nutritional biochemistry.

[22]  Xiangqun Fang,et al.  MicroRNA-144 regulates proliferation, invasion, and apoptosis of cells in malignant solitary pulmonary nodule via zinc finger E-box-binding homeobox 1. , 2015, International journal of clinical and experimental pathology.

[23]  Stefan Sleijfer,et al.  MicroRNA expression profiles distinguish liposarcoma subtypes and implicate miR‐145 and miR‐451 as tumor suppressors , 2014, International journal of cancer.

[24]  Christina Backes,et al.  Bias in High-Throughput Analysis of miRNAs and Implications for Biomarker Studies. , 2016, Analytical chemistry.

[25]  Muneesh Tewari,et al.  Quantitative and stoichiometric analysis of the microRNA content of exosomes , 2014, Proceedings of the National Academy of Sciences.

[26]  O. Kent,et al.  MicroRNA profiling of diverse endothelial cell types , 2011, BMC Medical Genomics.

[27]  Andrew Fire,et al.  Distinct Populations of Primary and Secondary Effectors During RNAi in C. elegans , 2007, Science.

[28]  Heidi J. Peltier,et al.  Normalization of microRNA expression levels in quantitative RT-PCR assays: identification of suitable reference RNA targets in normal and cancerous human solid tissues. , 2008, RNA.

[29]  Akhilesh Pandey,et al.  miRge - A Multiplexed Method of Processing Small RNA-Seq Data to Determine MicroRNA Entropy , 2015, PloS one.

[30]  Zefeng Zhang,et al.  RETRACTED ARTICLE: Regulation of activating protein-4-associated metastases of non-small cell lung cancer cells by miR-144 , 2016, Tumor Biology.

[31]  Juan Li,et al.  MiR-144 Inhibits Proliferation and Induces Apoptosis and Autophagy in Lung Cancer Cells by Targeting TIGAR , 2015, Cellular Physiology and Biochemistry.

[32]  Lin He,et al.  miR-34a Blocks Osteoporosis and Bone Metastasis by Inhibiting Osteoclastogenesis and Tgif2 , 2014, Nature.

[33]  Shu Liu,et al.  Repression of the miR-143/145 cluster by oncogenic Ras initiates a tumor-promoting feed-forward pathway. , 2010, Genes & development.

[34]  Ron Milo,et al.  Are We Really Vastly Outnumbered? Revisiting the Ratio of Bacterial to Host Cells in Humans , 2016, Cell.

[35]  Yukio Kitade,et al.  Downregulation of microRNAs‐143 and ‐145 in B‐cell malignancies , 2007, Cancer science.

[36]  B. Gelman,et al.  Deregulation of microRNAs by HIV-1 Vpr Protein Leads to the Development of Neurocognitive Disorders* , 2011, The Journal of Biological Chemistry.

[37]  Yan Zhang,et al.  MicroRNA-451 inhibits growth of human colorectal carcinoma cells via downregulation of Pi3k/Akt pathway. , 2013, Asian Pacific journal of cancer prevention : APJCP.

[38]  Min Wei,et al.  MicroRNA-144 suppresses osteosarcoma growth and metastasis by targeting ROCK1 and ROCK2 , 2015, Oncotarget.

[39]  S. Ingvarsson,et al.  MicroRNA-451 suppresses tumor cell growth by down-regulating IL6R gene expression. , 2014, Cancer epidemiology.

[40]  S. Le Guillou,et al.  No effect of an elevated miR-30b level in mouse milk on its level in pup tissues , 2015, RNA biology.

[41]  A. Fire,et al.  Specific interference by ingested dsRNA , 1998, Nature.

[42]  R. Sachidanandam,et al.  High-throughput assessment of microRNA activity and function using microRNA sensor and decoy libraries , 2012, Nature Methods.

[43]  G. Schratt microRNAs at the synapse , 2011, Nature Reviews Cancer.

[44]  J. McCarthy MicroRNA-206: the skeletal muscle-specific myomiR. , 2008, Biochimica et biophysica acta.

[45]  C. Bracken,et al.  On Measuring miRNAs after Transient Transfection of Mimics or Antisense Inhibitors , 2013, PloS one.

[46]  Isidore Rigoutsos,et al.  Interactive exploration of RNA22 microRNA target predictions , 2012, Bioinform..

[47]  C. Xiao,et al.  Transfection of microRNA Mimics Should Be Used with Caution , 2015, Front. Genet..

[48]  J. Lykke-Andersen,et al.  The control of mRNA decapping and P-body formation. , 2008, Molecular cell.

[49]  Robert A. Smith,et al.  Deregulation of miR-126 expression in colorectal cancer pathogenesis and its clinical significance. , 2015, Experimental cell research.

[50]  Jialing Huang,et al.  Cellular microRNAs contribute to HIV-1 latency in resting primary CD4+ T lymphocytes , 2007, Nature Medicine.

[51]  E. Kroh,et al.  Blood Cell Origin of Circulating MicroRNAs: A Cautionary Note for Cancer Biomarker Studies , 2011, Cancer Prevention Research.

[52]  Claus Lindbjerg Andersen,et al.  Normalization of Real-Time Quantitative Reverse Transcription-PCR Data: A Model-Based Variance Estimation Approach to Identify Genes Suited for Normalization, Applied to Bladder and Colon Cancer Data Sets , 2004, Cancer Research.

[53]  E. H. Feinberg,et al.  Transport of dsRNA into Cells by the Transmembrane Protein SID-1 , 2003, Science.

[54]  Anton J. Enright,et al.  Chimira: analysis of small RNA sequencing data and microRNA modifications , 2015, Bioinform..

[55]  Ryan T Fuchs,et al.  Bias in Ligation-Based Small RNA Sequencing Library Construction Is Determined by Adaptor and RNA Structure , 2015, PloS one.

[56]  C. Caldas,et al.  Double-stranded microRNA mimics can induce length- and passenger strand–dependent effects in a cell type–specific manner , 2016, RNA.

[57]  John McAnally,et al.  The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis. , 2008, Developmental cell.

[58]  Tengteng Zhang,et al.  MicroRNA-126 regulates migration and invasion of gastric cancer by targeting CADM1. , 2015, International journal of clinical and experimental pathology.

[59]  Klaus Pantel,et al.  Data Normalization Strategies for MicroRNA Quantification. , 2015, Clinical chemistry.

[60]  E. Wentzel,et al.  miR-21: an androgen receptor-regulated microRNA that promotes hormone-dependent and hormone-independent prostate cancer growth. , 2009, Cancer research.

[61]  Duan Ma,et al.  The cell growth suppressor, mir-126, targets IRS-1. , 2008, Biochemical and biophysical research communications.

[62]  Xuan Yang,et al.  MiR-451 inhibits proliferation of esophageal carcinoma cell line EC9706 by targeting CDKN2D and MAP3K1. , 2015, World journal of gastroenterology.

[63]  D. Kreisel,et al.  MicroRNA-144 dysregulates the transforming growth factor-β signaling cascade and contributes to the development of bronchiolitis obliterans syndrome after human lung transplantation. , 2015, The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation.

[64]  K. Witwer XenomiRs and miRNA homeostasis in health and disease , 2012, RNA biology.

[65]  X. Chen,et al.  Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA , 2011, Cell Research.

[66]  S. Thibodeau,et al.  Characterization of human plasma-derived exosomal RNAs by deep sequencing , 2013, BMC Genomics.

[67]  Jeffrey E. Thatcher,et al.  Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis , 2008, Proceedings of the National Academy of Sciences.

[68]  J. Tosar,et al.  Assessment of small RNA sorting into different extracellular fractions revealed by high-throughput sequencing of breast cell lines , 2015, Nucleic acids research.

[69]  V. Mandys,et al.  Small nucleolar RNA U91 is a new internal control for accurate microRNAs quantification in pancreatic cancer , 2015, BMC Cancer.

[70]  K. Witwer,et al.  Validated MicroRNA Target Databases: An Evaluation , 2015, Drug development research.

[71]  Yuanji Zhang,et al.  Analysis of plant-derived miRNAs in animal small RNA datasets , 2012, BMC Genomics.

[72]  Toby C. Cornish,et al.  Lessons from miR-143/145: the importance of cell-type localization of miRNAs , 2014, Nucleic acids research.

[73]  Johnny J. He,et al.  HIV-1 Tat Protein Promotes Neuronal Dysfunction through Disruption of MicroRNAs* , 2011, The Journal of Biological Chemistry.

[74]  J. Ioannidis,et al.  Design and Analysis for Studying microRNAs in Human Disease: A Primer on -Omic Technologies. , 2014, American journal of epidemiology.

[75]  Jianfang Li,et al.  miR-126 functions as a tumour suppressor in human gastric cancer. , 2010, Cancer letters.

[76]  K. Zen,et al.  Role of miR‐150‐targeting c‐Myb in colonic epithelial disruption during dextran sulphate sodium‐induced murine experimental colitis and human ulcerative colitis , 2011, The Journal of pathology.

[77]  J. Xiang,et al.  miR-451: potential role as tumor suppressor of human hepatoma cell growth and invasion. , 2014, International journal of oncology.

[78]  Jianyi Li,et al.  Influence of MiR-451 on Drug Resistances of Paclitaxel-Resistant Breast Cancer Cell Line , 2015, Medical science monitor : international medical journal of experimental and clinical research.

[79]  Mateusz Kuzak,et al.  Improving small RNA-seq by using a synthetic spike-in set for size-range quality control together with a set for data normalization , 2015, Nucleic acids research.

[80]  Xiuping Liu,et al.  Role of MicroRNA miR-27a and miR-451 in the regulation of MDR1/P-glycoprotein expression in human cancer cells. , 2008, Biochemical pharmacology.

[81]  K. Hirschi,et al.  Transfer and functional consequences of dietary microRNAs in vertebrates: Concepts in search of corroboration , 2014, BioEssays : news and reviews in molecular, cellular and developmental biology.

[82]  Wei R. Chen,et al.  miR-451 inhibits cell proliferation in human hepatocellular carcinoma through direct suppression of IKK-β. , 2013, Carcinogenesis.

[83]  Xiaofei Zheng,et al.  miR‐486 regulates metastasis and chemosensitivity in hepatocellular carcinoma by targeting CLDN10 and CITRON , 2015, Hepatology research : the official journal of the Japan Society of Hepatology.

[84]  E. H. Feinberg,et al.  Caenorhabditis elegans SID-2 is required for environmental RNA interference , 2007, Proceedings of the National Academy of Sciences.

[85]  M. Laakso,et al.  Genetic regulation of human adipose microRNA expression and its consequences for metabolic traits. , 2013, Human molecular genetics.

[86]  Jan Krüger,et al.  RNAhybrid: microRNA target prediction easy, fast and flexible , 2006, Nucleic Acids Res..

[87]  Vikram Agarwal,et al.  Assessing the ceRNA hypothesis with quantitative measurements of miRNA and target abundance. , 2014, Molecular cell.

[88]  David J. Galas,et al.  Comparing the MicroRNA Spectrum between Serum and Plasma , 2012, PloS one.

[89]  M. Halushka,et al.  Extracellular vesicle microRNA transfer in cardiovascular disease. , 2015, Cardiovascular pathology : the official journal of the Society for Cardiovascular Pathology.

[90]  Raffaella Casadei,et al.  An estimation of the number of cells in the human body , 2013, Annals of human biology.

[91]  N. Seki,et al.  Tumour-suppressive microRNA-144-5p directly targets CCNE1/2 as potential prognostic markers in bladder cancer , 2015, British Journal of Cancer.

[92]  J. Lötvall,et al.  Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells , 2007, Nature Cell Biology.

[93]  Shang‐dang Cai,et al.  MicroRNA-144 inhibits migration and proliferation in rectal cancer by downregulating ROCK-1 , 2015, Molecular medicine reports.

[94]  Juan Zhao,et al.  MiR-451, a potential prognostic biomarker and tumor suppressor for gastric cancer. , 2015, International journal of clinical and experimental pathology.

[95]  Daniel B. Martin,et al.  Circulating microRNAs as stable blood-based markers for cancer detection , 2008, Proceedings of the National Academy of Sciences.

[96]  Yuanji Zhang,et al.  Lack of detectable oral bioavailability of plant microRNAs after feeding in mice , 2013, Nature Biotechnology.

[97]  T. Duchaine,et al.  On the availability of microRNA-induced silencing complexes, saturation of microRNA-binding sites and stoichiometry , 2015, Nucleic acids research.

[98]  Praveen Sethupathy,et al.  Addressing Bias in Small RNA Library Preparation for Sequencing: A New Protocol Recovers MicroRNAs that Evade Capture by Current Methods , 2015, Front. Genet..

[99]  Xiwei Wu,et al.  Cross-kingdom inhibition of breast cancer growth by plant miR159 , 2016, Cell Research.

[100]  Toby C. Cornish,et al.  A Critical Evaluation of microRNA Biomarkers in Non-Neoplastic Disease , 2014, PloS one.

[101]  Huili Wang,et al.  Suppression of liver receptor homolog-1 by microRNA-451 represses the proliferation of osteosarcoma cells. , 2015, Biochemical and biophysical research communications.

[102]  Clotilde Théry,et al.  Communication by Extracellular Vesicles: Where We Are and Where We Need to Go , 2016, Cell.

[103]  D. Tollervey,et al.  Mapping the miRNA interactome by cross-linking ligation and sequencing of hybrids (CLASH) , 2014, Nature Protocols.

[104]  Y. Tu,et al.  MicroRNA-144 suppresses tumorigenesis and tumor progression of astrocytoma by targeting EZH2. , 2015, Human pathology.

[105]  Zheng Zhang,et al.  MiR-451 Suppresses Cell Proliferation and Metastasis in A549 Lung Cancer Cells , 2014, Molecular Biotechnology.

[106]  S. Chan,et al.  Ineffective delivery of diet-derived microRNAs to recipient animal organisms , 2013, RNA biology.

[107]  A. Maitra,et al.  An Essential Mesenchymal Function for miR-143/145 in Intestinal Epithelial Regeneration , 2014, Cell.

[108]  D. Pegtel,et al.  Extracellular Vesicles Exploit Viral Entry Routes for Cargo Delivery , 2016, Microbiology and Molecular Reviews.

[109]  P. Pandolfi,et al.  A ceRNA Hypothesis: The Rosetta Stone of a Hidden RNA Language? , 2011, Cell.

[110]  Hervé Seitz,et al.  Redefining MicroRNA Targets , 2009, Current Biology.

[111]  W. Gerald,et al.  Endogenous human microRNAs that suppress breast cancer metastasis , 2008, Nature.

[112]  Thean-Hock Tang,et al.  Biases in small RNA deep sequencing data , 2013, Nucleic acids research.

[113]  W. De,et al.  MicroRNA-451: epithelial-mesenchymal transition inhibitor and prognostic biomarker of hepatocelluar carcinoma , 2015, Oncotarget.

[114]  E. Sverdlov Amedeo Avogadro's cry: What is 1 µg of exosomes? , 2012, BioEssays : news and reviews in molecular, cellular and developmental biology.

[115]  Xianghong Zhang,et al.  Pim-1 kinase is a target of miR-486-5p and eukaryotic translation initiation factor 4E, and plays a critical role in lung cancer , 2014, Molecular Cancer.

[116]  Wei Huang,et al.  MiR-451 inhibits cell growth and invasion by targeting CXCL16 and is associated with prognosis of osteosarcoma patients , 2015, Tumor Biology.

[117]  Wei Wang,et al.  MicroRNA-34b and MicroRNA-34c are targets of p53 and cooperate in control of cell proliferation and adhesion-independent growth. , 2007, Cancer research.

[118]  Yi Cao,et al.  Down-regulation of microRNA-144 in air pollution-related lung cancer , 2015, Scientific Reports.

[119]  M. Stoffel,et al.  Uptake and Function Studies of Maternal Milk-derived MicroRNAs* , 2015, The Journal of Biological Chemistry.

[120]  Hugo Naya,et al.  Mining of public sequencing databases supports a non-dietary origin for putative foreign miRNAs: underestimated effects of contamination in NGS , 2014, RNA.

[121]  W. Ritchie,et al.  Predicting microRNA targets and functions: traps for the unwary , 2009, Nature Methods.

[122]  Ling Qiu,et al.  MicroRNA-144 affects radiotherapy sensitivity by promoting proliferation, migration and invasion of breast cancer cells. , 2015, Oncology reports.

[123]  C. Blenkiron Uptake of dietary milk miRNAs by adult humans : a validation study , 2017 .

[124]  D. Bartel,et al.  Predicting effective microRNA target sites in mammalian mRNAs , 2015, eLife.