miRNAs can be generally associated with human pathologies as exemplified for miR-144*

BMC Medicine miRNAs can be generally associated with human pathologies as exemplified for miR-144* Andreas Keller 0 Petra Leidinger Britta Vogel Christina Backes 0 Abdou ElSharawy Valentina Galata 0 Sabine C Mueller 0 Sabine Marquart Michael G Schrauder Reiner Strick Andrea Bauer Jrg Wischhusen Markus Beier Jochen Kohlhaas Hugo A Katus Jrg Hoheisel Andre Franke Benjamin Meder Eckart Meese 0 Chair for Clinical Bioinformatics, Saarland University , Saarbrucken , Germany Background: miRNA profiles are promising biomarker candidates for a manifold of human pathologies, opening new avenues for diagnosis and prognosis. Beyond studies that describe miRNAs frequently as markers for specific traits, we asked whether a general pattern for miRNAs across many diseases exists. Methods: We evaluated genome-wide circulating profiles of 1,049 patients suffering from 19 different cancer and non-cancer diseases as well as unaffected controls. The results were validated on 319 individuals using qRT-PCR. Results: We discovered 34 miRNAs with strong disease association. Among those, we found substantially decreased levels of hsa-miR-144* and hsa-miR-20b with AUC of 0.751 (95% CI: 0.703-0.799), respectively. We also discovered a set of miRNAs, including hsa-miR-155*, as rather stable markers, offering reasonable control miRNAs for future studies. The strong downregulation of hsa-miR-144* and the less variable pattern of hsa-miR-155* has been validated in a cohort of 319 samples in three different centers. Here, breast cancer as an additional disease phenotype not included in the screening phase has been included as the 20th trait. Conclusions: Our study on 1,368 patients including 1,049 genome-wide miRNA profiles and 319 qRT-PCR validations further underscores the high potential of specific blood-borne miRNA patterns as molecular biomarkers. Importantly, we highlight 34 miRNAs that are generally dysregulated in human pathologies. Although these markers are not specific to certain diseases they may add to the diagnosis in combination with other markers, building a specific signature. Besides these dysregulated miRNAs, we propose a set of constant miRNAs that may be used as control markers. Bioinformatics; Biomarker; Microarray; miRNA - Background In the past decade, non-coding miRNAs have aroused scientists interest and their exploration has revolutionized biology. Since the first miRNA was discovered in Caenorhabditis elegans in 1993 [1], an increasing number of miRNAs for various species have been reported. Currently, release 20 of the miRBase [2,3] contains 24,521 entries representing hairpin precursor miRNAs, expressing 30,424 mature miRNA products in 206 species. For Homo sapiens, more than 2,500 different mature miRNAs are currently included in this database. The small non-coding miRNAs are known to be involved in crucial biological processes such as proliferation, apoptosis, differentiation, or development [4-6]. More than 50% of all genes in the human genome are known to be miRNA targets and, thus, miRNAs are involved in the regulation of a manifold of metabolic and regulatory pathways such that now the integrative network analysis of miRNAs and mRNAs becomes more and more possible [7-9]. Hence, abnormal miRNA profiles have been associated with many human pathogenic processes as shown by many studies that focused on tissue-derived miRNA profiles (e.g., from patients with lung cancer [10], breast cancer [11], or glioblastoma [12]). Since these small nucleic acids excel in their high stability, they have become even more attractive as biomarker candidates. This also underlines the potential of miRNA biomarkers derived from peripheral blood for diagnostic purposes. Many groups investigated circulating miRNA profiles from serum for various diseases (non-ischemic systolic heart failure [13], pulmonary tuberculosis [14], non-small-cell lung cancer [15,16], breast cancer [17], prostate cancer [18], or ovarian cancer [19]), whereas we and others developed standardized operating procedures for measuring miRNA profiles from whole peripheral blood (myocardial infarction [20], lung cancer [21], multiple sclerosis [22,23], melanoma [24], ovarian cancer [25], chronic obstructive pulmonary disease [26], glioblastoma [27], and Alzheimer disease [28]). In the present meta-analysis, we analyzed a total of 848 miRNAs in 1,049 samples (containing the 454 samples published in our previous study [29]) measured from whole blood collected in PAXgene blood tubes. The investigated cohort includes healthy controls as well as patients diagnosed with one of 19 diseases of different International Classification of Diseases (ICD)-10 classes (10 cancer entities and 9 non-cancer diseases; details on the different cohort sizes are presented in Table 1). Our results provide a comprehensive overview of the human disease miRNome. By using this rich data source, we aimed at identifying miRNA profiles representative for a general disease state, and to identify miRNA signatures that are suited to discriminate different diseases from controls and from each other. Methods Blood samples and groups The blood samples were collected and processed from nine different institutions (Table 1). Five centers provided samples from individuals with disease as well as controls. Blood was collected in PAXgene Blood RNA Table 1 Cohorts with International Classification of Diseases (ICD)-10 code and cohort sizes Pancreatic ductal adenocarcinoma Acute myocardial infarction Non-ischemic systolic heart failure Chronic obstructive pulmonary disease Benign prostate hyperplasia Julius-Maximilians-University Wuerzburg Christian-Albrechts-University Kiel Christian-Albrechts-University Kiel Julius-Maximilians-University Wuerzburg Albrecht Ludwigs University, Freiburg Christian-Albrechts-University Kiel tubes (Becton Dickinson). All blood donors participating in this study gave their informed consent and local ethics committees (Ethics Commission at the FriedrichAlexander University Erlangen-Nrnberg Medical School; Ethics Commission of the Christian-Albrechts-University Kiel; Ethics Committee at the University of Wrzburg Medical School; rztekammer des Saarlandes; Ethics Committee Heidelberg University) approved the studies. An overview of all patients is presented in Additional file 1: Table S1. Selected diseases/traits have been grouped together as others. This includes patients with unclear diagnosis, e.g., patients that have either a pancreatic cancer or pancreatitis or patients with prostate cancer or benign prostate hyperplasia. The others group also contains some very small cohorts, e.g., 6 samples with atopic dermatitis. One group has been left out of the pairwise comparisons, namely the 15 long-lived individuals that show a substantial age bias since these would potentially bias either the control or disease profiles. miRNA extraction and microarray screening miRNA extraction and microarray measurement have been carried out as p (...truncated)