microRNAs In Biofluids Research Articles

Tear Film MicroRNAs as Potential Biomarkers: A Review.

MicroRNAs are non-coding RNAs that serve as regulatory molecules in a variety of pathways such as inflammation, metabolism, homeostasis, cell machinery, and development. With the progression of sequencing methods and modern bioinformatics tools, novel roles of microRNAs in regulatory mechanisms and pathophysiological states continue to expand. Advances in detection methods have further enabled larger adoption of studies utilizing minimal sample volumes, allowing the analysis of microRNAs in low-volume biofluids, such as the aqueous humor and tear fluid. The reported abundance of extracellular microRNAs in these biofluids has prompted studies to explore their biomarker potential. This review compiles the current literature reporting microRNAs in human tear fluid and their association with ocular diseases including dry eye disease, Sjögren's syndrome, keratitis, vernal keratoconjunctivitis, glaucoma, diabetic macular edema, and diabetic retinopathy, as well as non-ocular diseases, including Alzheimer's and breast cancer. We also summarize the known roles of these microRNAs and shed light on the future progression of this field.

Urinary microRNAs as non-invasive biomarkers for toxic acute kidney injury in humans

MicroRNAs in biofluids are potential biomarkers for detecting kidney and other organ injuries. We profiled microRNAs in urine samples from patients with Russell’s viper envenoming or acute self-poisoning following paraquat, glyphosate, or oxalic acid [with and without acute kidney injury (AKI)] and on healthy controls. Discovery analysis profiled for 754 microRNAs using TaqMan OpenArray qPCR with three patients per group (12 samples in each toxic agent). From these, 53 microRNAs were selected and validated in a larger cohort of patients (Russell’s viper envenoming = 53, paraquat = 51, glyphosate = 51, oxalic acid = 40) and 27 healthy controls. Urinary microRNAs had significantly higher expression in patients poisoned/envenomed by different nephrotoxic agents in both discovery and validation cohorts. Seven microRNAs discriminated severe AKI patients from no AKI for all four nephrotoxic agents. Four microRNAs (miR-30a-3p, miR-30a-5p, miR-92a, and miR-204) had > 17 fold change (p < 0.0001) and receiver operator characteristics area-under-curve (ROC-AUC) > 0.72. Pathway analysis of target mRNAs of these differentially expressed microRNAs showed association with the regulation of different nephrotoxic signaling pathways. In conclusion, human urinary microRNAs could identify toxic AKI early after acute injury. These urinary microRNAs have potential clinical application as early non-invasive diagnostic AKI biomarkers.

Potential role of MicroRNA as a diagnostic tool in the detection of bovine mastitis

Bovine mastitis is a major health problem that affects dairy cows and has a negative impact on milk production. The presence of microRNAs in biofluids, such as blood and milk, could play a pivotal role in the detection of bovine mastitis. The purpose of the current study was to determine the levels of microRNA gene expression in milk, in combination with other reported mastitis indicators, as a biomarker of bovine mastitis. Milk samples (n = 171) were obtained from 113 dairy cows with known disease status (i.e., healthy; n = 23 cows, subclinical mastitis; n = 45 cows, or clinical mastitis; n = 45 cows) and analyzed for the presence of MIR24-2, MIR29B-2, MIR146A, MIR148A, MIR155, MIR181A1, MIR184, and MIR223 expression using the real-time PCR (qPCR) method. The expression data were then utilized in the creation of receiver operator characteristic curves (ROC) and further analyzed by the machine learning (ML) methods. MIR29B-2, MIR146A, MIR148A, and MIR155 expression levels differed significantly among the three groups. These potential microRNA biomarkers of mastitis exhibited high sensitivity and specificity. Next, we applied ML algorithm, specifically, a decision tree (DT) model to predict the status of milk based on MIR29B-2 and MIR146A expression levels. The results suggested that MIR29B-2, when used in combination with the California mastitis test (CMT) and days in milk (DIM) data, was applicable for screening and classification of milk samples from cows as healthy, subclinical mastitis, or mastitis. MIR29B-2 appears to have sufficient discriminatory power to enable it to be utilized as a biomarker in cases where the status of a milk sample cannot be determined based on CMT results.

Genome-wide microRNA profiling of plasma from three different animal models identifies biomarkers of temporal lobe epilepsy

Epilepsy diagnosis is complex, requires a team of specialists and relies on in-depth patient and family history, MRI-imaging and EEG monitoring. There is therefore an unmet clinical need for a non-invasive, molecular-based, biomarker to either predict the development of epilepsy or diagnose a patient with epilepsy who may not have had a witnessed seizure. Recent studies have demonstrated a role for microRNAs in the pathogenesis of epilepsy. MicroRNAs are short non-coding RNA molecules which negatively regulate gene expression, exerting profound influence on target pathways and cellular processes. The presence of microRNAs in biofluids, ease of detection, resistance to degradation and functional role in epilepsy render them excellent candidate biomarkers.Here we performed the first multi-model, genome-wide profiling of plasma microRNAs during epileptogenesis and in chronic temporal lobe epilepsy animals. From video-EEG monitored rats and mice we serially sampled blood samples and identified a set of dysregulated microRNAs comprising increased miR-93-5p, miR-142-5p, miR-182-5p, miR-199a-3p and decreased miR-574-3p during one or both phases. Validation studies found miR-93-5p, miR-199a-3p and miR-574-3p were also dysregulated in plasma from patients with intractable temporal lobe epilepsy. Treatment of mice with common anti-epileptic drugs did not alter the expression levels of any of the five miRNAs identified, however administration of an anti-epileptogenic microRNA treatment prevented dysregulation of several of these miRNAs. The miRNAs were detected within the Argonuate2-RISC complex from both neurons and microglia indicating these miRNA biomarker candidates can likely be traced back to specific brain cell types.The current studies identify additional circulating microRNA biomarkers of experimental and human epilepsy which may support diagnosis of temporal lobe epilepsy via a quick, cost-effective rapid molecular-based test.

SAT-132 CIRCULATING MICRORNAS DETECT ACUTE KIDNEY INJURY FOLLOWING INGESTION OF POTASSIUM PERMANGANATE AND OXALIC ACID

Early detection of acute kidney injury (AKI) is difficult due to the lack of sensitive and specific biomarkers. MicroRNAs are stable biomarkers, which are released following cellular injury. Local and systemic effects of tissue injury can be captured via efficient detection of microRNAs in bio-fluids. This study aims to identify a signature of serum microRNAs as early biomarkers of AKI in patients following poisoning with oxalic acid and potassium permanganate.

New Platform for the Direct Profiling of microRNAs in Biofluids.

Circulating microRNAs have been identified as potential biomarkers for early detection, prognosis, and prediction of several diseases. Their use in clinical diagnostics has been limited by the lack of suitable detection techniques. Most of the current technologies suffer from requiring complex protocols, not yet able to deliver robust and cost-effective assays in the field of clinical diagnostics. In this work, we report the development of a breakthrough platform for profiling circulating microRNAs. The platform comprises a novel silicon photomultiplier-based reader in conjunction with a chemical-based method for nucleic acid detection. Accurate microRNAs profiling without extraction, pre-amplification, or pre-labeling of the target is now achievable. We designed and synthesized a set of reagents that combined the chemical-based method with a chemiluminescent reaction. The signals generated were read outusing a novel, compact silicon photomultiplier-based reader. The platform sensitivity was determined by measuring known concentrations of hsa-miR-21-5p spike-ins. The limit of detection was calculated as 4.7 pmol/L. The platform was also successfully used to directly detect hsa-miR-21-5p in eight non-small cell lung cancer plasma samples. Levels of plasma hsa-miR-21-5p expression were also measured via TaqMan RT-qPCR. The successful integration of theunique chemical-based method for nucleic acid detection with the novel silicon photomultiplier-based reader created an innovative product (ODG platform) with diagnostic utility, for the direct qualitative and quantitative analysis of microRNA biomarkers in biological fluids.

MiR-21-5p, miR-141-3p, and miR-205-5p levels in urine-promising biomarkers for the identification of prostate and bladder cancer.

Early detection of cancers improves patients' survival and decreases the treatment cost. Unfortunately, the current methods for diagnosis of bladder and prostate cancers, two most common urothelial malignancies, suffer from a low sensitivity and specificity. MicroRNAs, as a group of endogenously produced non-coding RNAs, regulate gene expression and their expression is observed to be altered in many cancers and cancer progression phenomena. The remarkable stability of microRNAs in biofluids and their unique expression pattern in different pathological conditions make them an appealing, noninvasive diagnostic method in cancer diagnosis. Our objective is to identify microRNAs as biomarkers in urine samples of bladder and prostate cancers to improve the existing diagnostic methods in this field. In this study, urine samples from 110 men with either bladder (n = 45) or prostate (n = 23) cancer, benign prostatic hyperplasia (n = 22) and healthy controls (n = 20) were collected. qPCR was used to evaluate the expression level of miR-21-5p, miR-141-3p, and miR-205-5p in these samples. The sensitivity and specificity of these microRNAs were determined using ROC curve analysis. The analysis of the data revealed that miR-21-5p, miR-141-3p, and miR-205-5p are differentially expressed in urine of bladder and prostate cancer patients. All these three microRNAs were upregulated in these samples and they were also able to differentiate benign prostatic hyperplasia from malignant cases. The statistical analyses revealed a good specificity for each individual microRNA. The results show that these three urine-based microRNAs might be a good choice to implement a specific and non-invasive diagnostic tool for bladder and prostate cancer. The expression pattern of all three microRNAs was particularly useful to distinguish benign and invasive tumors in prostate cases. From the patients' perspective the improvement of the diagnostic situation is awaited eagerly.

MP18-06 VALUE OF CIRCULATING MICRORNAS AS PROGNOSTIC BIOMARKERS IN UPPER TRACT UROTHELIAL CARCINOMA

You have accessJournal of UrologyBladder Cancer: Upper Tract Transitional Cell Carcinoma I1 Apr 2018MP18-06 VALUE OF CIRCULATING MICRORNAS AS PROGNOSTIC BIOMARKERS IN UPPER TRACT UROTHELIAL CARCINOMA Ruth Montalbo, Laura Izquierdo, Mercedes Ingelmo-Torres, Juan Jose Lozano, Noelia Ventura, Antonio Alcaraz, and Lourdes Mengual Ruth MontalboRuth Montalbo More articles by this author , Laura IzquierdoLaura Izquierdo More articles by this author , Mercedes Ingelmo-TorresMercedes Ingelmo-Torres More articles by this author , Juan Jose LozanoJuan Jose Lozano More articles by this author , Noelia VenturaNoelia Ventura More articles by this author , Antonio AlcarazAntonio Alcaraz More articles by this author , and Lourdes MengualLourdes Mengual More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.582AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Upper tract urothelial carcinoma (UTUC) represents an aggressive disease with high recurrence and progression rates. The 5-year specific survival for these tumors is < 50% and 60% of them are invasive at the moment of diagnosis. Several studies have investigated the prognostic impact of various tissue-based markers that are related to different cellular processes. However, none of them has fulfilled the clinical and statistical criteria necessary to support their introduction in daily clinical decision making. In this study we aim to identify microRNA expression profiles in serum as a non-invasive prognostic tool for UTUC patients. METHODS Prospective study of serum samples from 33 UTUC patients (13 progressing and 20 non-progressing), who underwent radical nephroureterectomy in Hospital Clinic of Barcelona, Spain. Total RNA was isolated and expression of 800 microRNAs was analyzed using nCounter® miRNA Expression Assays (NanoString Technologies). Expression values of the 800 microRNAs were evaluated. The study was divided into an initial screening phase (n=12) and a validation phase (n=21). RESULTS Median age (range) of the cohort is 70 years (50-90). The median follow-up (range) was 42 months (9-100 months). Initial screening expression analysis identified 38 microRNA differentially expressed between the progressing and non-progressing UTUC patients (p<0.05). Of these, 9 were up-regulated and 29 were down-regulated. The validation phase, in an independent set of UTUC patients, demonstrated 18 microRNAs remaining differentially expressed between the two groups (8 progressing and 13 non-progressing). All validated microRNAs were down-regulated in progressing patients’ expression profiles. Survival models were used in this set of validated microRNAs. Cox Regression analysis showed miR-151b and stage as significant prognostic factors for tumor progression (p=0.000 and p=0.006 respectively) and cancer specific survival (p=0.000 and p=0.003 respectively). Finally, survival curves revealed that miR-151b is a statistically significant prognostic factor and discriminates between two groups of UTUC patients for progression prognosis (p=0.006) and for cancer specific survival (p=0.034). CONCLUSIONS Serum UTUC miRNA profiles can discriminate progressing versus non-progressing tumors. Further validation of these microRNAs in a larger cohort of patients is needed. This is the first study analyzing serum samples from UTUC patients. Detection of microRNAs in biofluids as serum could be a promising non-invasive tool to monitor progression in UTUC. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e215 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Ruth Montalbo More articles by this author Laura Izquierdo More articles by this author Mercedes Ingelmo-Torres More articles by this author Juan Jose Lozano More articles by this author Noelia Ventura More articles by this author Antonio Alcaraz More articles by this author Lourdes Mengual More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MicroRNAs in biofluids are novel tools for bladder cancer screening.

MicroRNAs (miRNAs) are short non-coding RNAs that play important roles in basic cellular processes, including differentiation, proliferation, apoptosis and autophagy. They are also involved in various stages of tumorigenesis and play key roles in bladder cancer initiation and progression. Notably, the altered expression of miRNAs in the tumors is reflected in body fluids, including blood and urine, which opens avenues for non-invasive diagnosis and prognosis. Many studies have demonstrated that epigenetic changes extensively alter tumoral microRNA expression. The high reproducibility, specificity and sensitivity of miRNA levels in body fluids suggest their potential use as biomarkers for cancer screening and diagnosis. For example, recent technological advances have made it possible to detect miRNAs in urine for bladder cancer screening. In this review, we focus mainly on the current knowledge and future challenges for incorporating miRNAs in body fluids, like urine and blood, for making clinical diagnoses and assessing prognoses in bladder cancer.

Quantitative RT-PCR for MicroRNAs in Biofluids.

MicroRNAs in biofluids hold great promise as minimally invasive diagnostic biomarkers for a wide range of diseases and biological processes. One of the most sensitive technologies for detection and measuring expression levels of microRNA is quantitative RT-PCR. However, quantification of microRNA in biofluid samples is challenging in many ways. Biofluids contain low levels of RNA and high levels of inhibitors of enzymatic processes like reverse transcription and PCR. Furthermore, biofluids are susceptible to many preanalytical variables. Here we describe procedures developed to address these challenges, which include highly sensitive and accurate microRNA detection methods, combined with optimized protocols for sample handling and preparation, and extensive quality control (QC) procedures.

Absolute Measurement of Cardiac Injury-Induced microRNAs in Biofluids across Multiple Test Sites.

Extracellular microRNAs (miRNAs) represent a promising new source of toxicity biomarkers that are sensitive indicators of site of tissue injury. In order to establish reliable approaches for use in biomarker validation studies, the HESI technical committee on genomics initiated a multi-site study to assess sources of variance associated with quantitating levels of cardiac injury induced miRNAs in biofluids using RT-qPCR. Samples were generated at a central site using a model of acute cardiac injury induced in male Wistar rats by 0.5 mg/kg isoproterenol. Biofluid samples were sent to 11 sites for measurement of 3 cardiac enriched miRNAs (miR-1-3p, miR-208a-3p, and miR-499-5p) and 1 miRNA abundant in blood (miR-16-5p) or urine (miR-192-5p) by absolute quantification using calibration curves of synthetic miRNAs. The samples included serum and plasma prepared from blood collected at 4 h, urine collected from 6 to 24 h, and plasma prepared from blood collected at 24 h post subcutaneous injection. A 3 parameter logistic model was utilized to fit the calibration curve data and estimate levels of miRNAs in biofluid samples by inverse prediction. Most sites observed increased circulating levels of miR-1-3p and miR-208a-3p at 4 and 24 h after isoproterenol treatment, with no difference seen between serum and plasma. The biological differences in miRNA levels and sample type dominated as sources of variance, along with outlying performance by a few sites. The standard protocol established in this study was successfully implemented across multiple sites and provides a benchmark method for further improvements in quantitative assays for circulating miRNAs.

Considerations for optimization of microRNA PCR assays for molecular diagnosis

ABSTRACTThe remarkable stability of microRNAs in biofluids underlies their potential as biomarkers, but their small size presents challenges for detection by RT-qPCR. The heterogeneity of microRNAs, with each one comprising a series of variants or ‘isomiRs’, adds additional complexity. Presented here are the key considerations for use of RT-qPCR to measure microRNAs and their isomiRs, with a focus on plasma. Modified nucleotides can be incorporated into primer sequences to enhance affinity and provide increased specificity and sensitivity for RT-qPCR assays. Approaches based upon polyA tailing and use of a common oligo(dT)-based reverse transcription oligonucleotide will detect most isomiRs. Conversely, stem-loop RT oligonucleotides and sequence specific probes can enable detection of specific isomiRs of interest. Next generation sequencing of all the products of a microRNA RT-PCR reaction is a promising new approach for both microRNA quantification and characterization.

ExcellmiRDB for translational genomics: a curated online resource for extracellular microRNAs.

A large number of studies have suggested extracellular microRNAs (microRNAs in biofluids) as potential noninvasive biomarkers for pathophysiological conditions such as cancer. However, reported differentially expressed signatures of extracellular miRNAs in diseases are not uniformly consistent among studies. Here, we present "ExcellmiRDB", a curated online database that provides integrated information about miRNAs levels in biofluids in a user-friendly way. Although many miRNA databases, including disease-oriented databases, have been launched before, the ExcellmiRDB is so far the only one specialized for storing curated data on miRNA levels in biofluid samples. At present, ExcellmiRDB has 2773 disease-extracellular miRNAs and 1108 biofluid-extracellular miRNAs relationships curated from 108 articles selected from more than 600 surveyed PubMed abstracts. Information about 992 miRNAs, 82 diseases, 21 biofluids, 8 species, 63 normalization reference genes, 5 techniques, 14 GEO profiles accession numbers, 7 human ethnic groups, and 18 compared clinical biomarkers have been provided in the database. A user can query ExcellmiRDB by selecting a disease or a miRNA or a biofluid. Additionally, the database provides two online network graphs to visualize and interact with the content of the database. The first network shows disease-extracellular miRNAs relationships, along with expression patterns and number of articles for a relationship. The second network visualizes biofluid-extracellular miRNAs relationships showing miRNAs spectrum across different types of biofluids. In conclusion, ExcellmiRDB is a new innovative resource for both academic and industrial researchers in translational omics who are developing miRNA biomarkers for noninvasive diagnostic or prognostic technologies. ExcellmiRDB is publicly available on www.excellmirdb.brfjaisalmer.com/.

Best practice for measurement of injury-associated microRNAs in biofluids

Best practice for measurement of injury-associated microRNAs in biofluids

Where are we in diabetic nephropathy

Several factors are now known to contribute to the development and progression of nephropathy, particularly in diabetes. In recent times, there has been surge of interest in the role of small noncoding RNA, with several reports focusing on the effects of microRNAs on their target genes that are of relevance to nephropathy. This review focuses on recent progress in this field. The list of microRNAs that have been identified to play a role in nephropathy continues to grow. Of particular interest is the fact that most microRNAs that are implicated in nephropathy are regulated by the profibrotic factor, transforming growth factor-β. Additionally, some recent studies have used the presence of microRNAs in biofluids as a source of potential biomarkers for many diseases, particularly in diabetic nephropathy. MicroRNAs hold much promise given their novelty, promiscuity and involvement in many biological and pathological processes. There are promising early signs of their potential as biomarkers as well as therapeutic targets.