miRNAs In Fluids Research Articles

Comparative study of RT-qPCR- and NGS- based platforms for circulating human microRNA biomarker detection

Abstract Background Circulating microRNAs (cmiRNAs) are emerging as promising non-invasive biomarkers in cancer for their diagnostic, prognostic and predictive potential. Several platforms have been developed to determine the presence of miRNAs in biological fluids, including specific focus panels. In this pilot study, we aimed at comparing different platforms for detection of cmiRNAs against the same cohort of plasma samples alongside RT-qPCR and NGS technologies. Methods RNA was isolated from 24 plasma samples obtained from lung cancer patients (n = 12, including one replicate) and healthy heavy smokers (n = 12, including two replicates). Six different platforms were used to detect human cmiRNAs. Five of them were high-throughput technologies, three RT-qPCR-based (TaqMan OpenArray and TaqMan OpenArray Advanced MicroRNA Panels by ThermoFisher; miRCURY LNA miRNA miRNome PCR panel by Qiagen) and two NGS-based (EdgeSeq miRNA byHTG and QiaSeq miRNA by Qiagen). The sixth platform, RT-qPCR-based and serum/plasma focused, was TaqMan Advanced miRNA Human Card by ThermoFisher. Samples were handled according to manufacturer’s instructions and data from each platform were analyzed using specific recommended thresholds. Results Selecting cmiRNAs yielding signals above background, we highlighted a subset commonly detected by all platforms. Strong concordance and correlation coefficients (>0.8) between the three replicate samples were obtained in most platforms, from independent RNA extractions. For each sample, the number of cmiRNAs detected ranged from 90 to 140. We found that inter-platform correlations were highest for platforms based on similar chemistry and assay design. Between lung cancer patients and healthy donors, few deregulated miRNAs could be however commonly detected by all platforms and studies are ongoing to elucidate their role. Conclusions This study is one of the first comparing RT-qPCR, NGS-based high-throughput and serum/plasma focused platforms for human cmiRNA biomarker detection. Advantages and limits of all platforms and data on intra- and inter- reproducibility will be presented to help investigators in identifying technologies better suited for cmiRNAs screening or validation in clinical cohorts. Legal entity responsible for the study The authors. Funding Italian Association for Cancer Research (AIRC, Special Programme 5 X 1000, ED No12162) to GS and MS and by Italian Ministry of Health (funds obtained through an Italian law that allows tax-payers to allocate the “5 × 1000” share of their payments to research) to LDC. Disclosure All authors have declared no conflicts of interest.

Distinct Profiles of Cell-Free MicroRNAs in Plasma of Veterans with Post-Traumatic Stress Disorder.

Dysregulation of circulating microRNAs (miRNAs) in body fluids has been reported in psychiatric disorders such as schizophrenia, bipolar disorder, major depressive disorder, and post-traumatic stress disorder (PTSD). Recent studies of various diseases showed that extracellular vesicles (EV) in body fluids can provide different spectra of circulating miRNAs and disease-associated signatures from whole fluid or EV-depleted fraction. However, the association of miRNAs in EVs to PTSD has not been studied. In this study, we performed a comprehensive profiling of miRNAs in whole plasma, extracellular vesicles (EV) and EV-depleted plasma (EVD) samples collected from combat veterans with PTSD and matched controls by utilizing a next-generation sequencing (NGS) platform. In total, 520 circulating miRNAs were quantified from 24 male Iraq and Afghanistan combat veterans with (n = 12) and without (n = 12) PTSD. The overall miRNA profiles in whole plasma, EV and EVD fractions were different and miRNAs affected by PTSD were also distinct in each sample type. The concentration changes of miR-203a-3p in EV and miR-339-5p in EVD were confirmed in an independent validation cohort that consisted of 20 veterans (10 with and 10 without PTSD) using qPCR. The target genes of these two miRNAs were involved in signaling pathways and comorbid conditions associated with PTSD (e.g., neurotransmitter systems such as dopaminergic and serotonergic signaling, inflammatory response, and cardiovascular diseases). Our findings suggest that PTSD may have different impacts on miRNAs encapsulated in vesicles and outside of vesicles. Further studies using larger samples are needed to evaluate the utility of these miRNAs as diagnostic biomarkers for PTSD.

PF539 MIR‐223 IS SUPPRESSED IN EXOSOMES OF PATIENTS WITH HIGH‐RISK MYELODYSPLASTIC SYNDROMES AND MAY BE A MARKER OF RESIDUAL GRANULOCYTIC‐MONOCYTIC DIFFERENTIATION

Background:Quantification of exosomal miRNAs in body fluids represents a prominent source of biomarkers for a variety of conditions. Several miRNAs have been described as potential predictors of severity and progression in neoplastic diseases, pointing to a class of less invasive and readily accessible prognostic tools. Among them, mir‐223 has been studied with contradictory findings: mir‐223 has been shown to play an important role in quimiorresistence in solid tumors (Cheng Q, et al., Mol Med Rep. 2017;15:597–604); however, suppression of mir‐223 in Acute Myeloid Leukemia (AML) is associated with lack of differentiation and adverse cytogenetic profile (Akhter A, et al., Appl Immunohistochem Mol Morphol, 2015;23:733–9).Aims:The aim of this study was to evaluate the expression of mir‐223 in exosomes extracted from serum of patients with myelodysplastic syndromes (MDS), AML and healthy blood donors and to explore the role of mir‐223 in the differentiation of human hematopoietic stem cells.Methods:Samples of venous blood were obtained from 14 patients with AML, 28 patients with MDS (prior to any treatment), and 19 healthy controls. Exosomes were isolated using the ExoQuick® precipitation solution; their concentration and size distribution were characterized by Nanoparticle Tracking Analysis (NanoSight, UK). Exosomal RNA was prepared using miRNeasy Micro Kit and submitted to reverse transcription with miScript II RT kit (QIAGEN, USA) and expression levels of mir‐223 and mir‐192 (endogenous control) were assessed by RT‐PCR using specific primers. CD34+ progenitors were purified from human umbilical cord blood using positive immunomagnetic selection and expanded in culture medium supplemented with G‐CSF, IL‐3 and SCF. The expression levels of mir‐223 were analyzed in cultured cells on days 0, 7, and 14; expression of CD34, CD14 and CD15 was verified by flow cytometry and morphologic changes were observed by examining Wright‐Giemsa‐stained cytospin smears.Results:The concentration of exosomes in serum samples ranged from 5.6 x 1011 to 3.3 x 1013 particles per μl of liquid suspension, with significantly lower concentrations in the patient samples in relation to healthy individuals (p = 0.004). A statistically significant decrease in the expression of mir‐223 was observed in exosomes from individuals with high‐risk MDS compared to healthy subjects (p = 0.002) and to patients with low‐risk MDS (p = 0.012). Interestingly, in the total group of patients, there was a statistically significant positive correlation between the expression of mir‐223 in the exosomes and countings of: total leukocytes (Spearman's r s = 0.37, p = 0.003), neutrophils (r s = 0.392, p = 0.002), monocytes (r s = 0.484, p < .001), lymphocytes (r s = 0.442, p < .001) and platelets (r s = 0.387, p = 0.002) in peripheral blood. During granulocytic‐monocytic differentiation of normal human CD34+ cells, we observed a 4‐fold increase in mir‐223 expression in cultured cells after 7 days, reaching almost 6‐fold increase after 14 days of culture, with granulocytic‐monocytic differentiation demonstrated by cytology and immunophenotyping with CD14 and CD15 markers.Summary/Conclusion:The results here obtained showed that levels of mir‐223 in peripheral blood exosomes are suppressed in patients with high‐risk MDS and correlate with cell counts in both MDS and AML. As mir‐223 levels progressively increase during normal granulocytic‐monocytic differentiation, we speculate that mir‐223 may represent a marker of residual differentiation in MDS and AML.

500-P: The Performance of Four Different Exosome Isolation Methods for Urinary Exosomal microRNA Profiling in Patients with Diabetic Kidney Disease

Both exosome and microRNAs (miRNAs) in biological fluids can be good biomarkers for pathologic conditions. Thus, urinary exosome miRNAs would be reasonable non-invasive tools for biomarker development for diabetic kidney disease. To be eligible for exosome miRNA biomarker development, the performance of exosome isolation methods for miRNA profiling need to be tested. In the present study, using urine samples from patients with type 2 diabetes, we compared four different exosome isolation methods for their performance in urine exosome isolation and detectable urinary exosome miRNA profiles. Four methods compared in the present study were ultracentrifugation (UC), size exclusion chromatography using qEV column (qEV), ExoQuick-TC (ExoQuick), and Amicon tube 100K (Amicon). Isolated exosomes were subjected to next generation sequencing (NGS) to profile miRNAs. The counts, RNA concentrations, and protein marker expressions of isolated urine exosome were variable between the extraction methods, with the highest exosome count with the qEV method. The number of miRNAs detected (read count ≥1) in 100 mL urine each were 314, 174, 91, and 130 from UC, qEV, ExoQuicK, and Amiconmethods, respectivley. And, 57 miRNAs were detected with all 4 methods concordantly. There were correlations in the levels of exosomal miRNAs between samples from UC, qEV, and ExoQuick methods, whereas exosome miRNA profiles from Amicon sample showed only a weak correlation with those from other methods. Our results suggest that exosome isolation by using UC or qEV method may be a better option among different isolation methods to profile urinary exosome miRNAs in diabetic kidney disease. However, ultrafiltration method using Amicon tube produced a different profile of miRNAs with other methods. Further studies are needed to characterize both exosome and their genetic materials from different extraction sources. Disclosure K. Lee: None. D. Lee: None. I. Park: None.

Regulation of Endothelial-to-Mesenchymal Transition by MicroRNAs in Chronic Allograft Dysfunction.

Fibrosis is a universal finding in chronic allograft dysfunction, and it is characterized by an accumulation of extracellular matrix. The precise source of the myofibroblasts responsible for matrix deposition is not understood, and pharmacological strategies for prevention or treatment of fibrosis remain limited. One source of myofibroblasts in fibrosis is an endothelial-to-mesenchymal transition (EndMT), a process first described in heart development and involving endothelial cells undergoing a phenotypic change to become more like mesenchymal cells. Recently, lineage tracing of endothelial cells in mouse models allowed studies of EndMT in vivo and reported 27% to 35% of myofibroblasts involved in cardiac fibrosis and 16% of isolated fibroblasts in bleomycin-induced pulmonary fibrosis to be of endothelial origin. Over the past decade, mature microRNAs (miRNAs) have increasingly been described as key regulators of biological processes through repression or degradation of targeted mRNA. The stability and abundance of miRNAs in body fluids make them attractive as potential biomarkers, and progress is being made in developing miRNA targeted therapeutics. In this review, we will discuss the evidence of miRNA regulation of EndMT from in vitro and in vivo studies and the potential relevance of this to heart, lung, and kidney allograft dysfunction.

Extracellular miRNAs as activators of innate immune receptors.

Since the discovery of the existence of microRNAs (miRNAs) in body fluids, the fascinating hypothesis that extracellular miRNAs may play a role in cell-to-cell signalling started to make its own way. In this review, we summarize the current knowledge that supports the role of miRNAs in the regulation of the immune response by an unconventional mechanism based on the activation of intracellular innate immune sensors of nucleic acids, namely the Toll-like receptors (TLRs). Such a mechanism of action has been now described to amplify and influence the pathogenesis of several inflammation-dependent pathological conditions, including cancer growth and metastasis, neurodegeneration, autoimmunity and cardiovascular diseases. The available data suggest that we have only begun to touch upon a complex system that is likely to involve many receptors and molecules. These findings may help to understand the pathogenesis of immune-mediated diseases and provide the basis for the identification of new potential therapeutic targets.

Quantitative and Specific Detection of Exosomal miRNAs for Accurate Diagnosis of Breast Cancer Using a Surface-Enhanced Raman Scattering Sensor Based on Plasmonic Head-Flocked Gold Nanopillars.

MicroRNAs in exosomes (exosomal miRNAs) have attracted increased attention as cancer biomarkers for early diagnosis and prognosis owing to their stability in body fluids. Since strong association exists between exosomal miRNA expression levels and breast cancer, the development of effective methods that can monitor exosomal miRNA expression both over broad concentration ranges and in ultralow amounts is critical. Here, a surface-enhanced Raman scattering (SERS)-based sensing platform is developed for the quantitative determination of exosomal miRNAs. Ultrasensitive exosomal miRNA detection with single-nucleotide specificity is obtained from enhanced SERS signals from a uniform plasmonic head-flocked gold nanopillar substrate, which generates multiple hotspots and enables hybridization between short oligonucleotides, i.e., miRNAs and locked nucleic acid probes. The proposed SERS sensor shows an extremely low detection limit without any amplification process, a wide dynamic range (1 am to 100 nm), multiplex sensing capability and sound miRNA recovery in serum. Furthermore, this sensor allows reliable observation of exosomal miRNA expression patterns from breast cancer cell lines and can discriminate breast cancer subtype based on the difference between these patterns. The results suggest that this sensor can be used for universal cancer diagnosis and further biomedical applications through the quantitative measurement of exosomal miRNAs in bodily fluids.

Characterization of circulating miRNA signature in water buffaloes (Bubalus bubalis) during Brucella abortus infection and evaluation as potential biomarkers for non-invasive diagnosis in vaginal fluid

Brucellosis is an infectious disease caused by bacteria from the Brucella genus that can be transmitted to humans through contact with infected animals or contaminated animal products. Brucellosis also causes financial losses in animal production. Ruminants are highly susceptible to brucellosis, and the causative agent water buffaloes (Bubalus bubalis) is Brucella abortus. Circulating microRNAs (miRNAs) are cropping up as promising biomarkers for several infectious diseases. The goals of this study were to characterize the serum miRNA signature associated with brucellosis in water buffaloes and investigate the miRNAs’ potential use as biomarkers in vaginal fluids. Next Generation Sequencing was used to assess miRNA expression profiles in Brucella-positive and Brucella-negative blood sera; dysregulated miRNAs in blood serum and vaginal fluids were validated using RT-qPCR. ROC curves were generated to evaluate the diagnostic value of miRNAs for Brucella. GO and KEGG pathway enrichment analyses were exploited to investigate the biological functions of dysregulated miRNAs. The results showed that 20 miRNAs were modulated, of which, 12 were upregulated and 8 were downregulated. These findings were corroborated by RT-qPCR, and ROC curves indicated that the miRNAs can serve as potential biomarkers for Brucella. GO and KEGG pathway analyses pointed out that some of these miRNAs are related to immune response and apoptosis. These results provided an overview of miRNA expression profiles and highlighted potential biomarkers for Brucella infection in water buffaloes. We also demonstrated the potential of vaginal fluids in studies involving microRNA detection. Further functional and mechanistic studies of these miRNAs may improve our understanding of the biological processes involved in Brucella infection and host immune response.

Circulating miRNAs as Diagnostic Biomarkers for Parkinson's Disease.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder worldwide. Its main neuropathological hallmarks are the degeneration of dopaminergic neurons in the substantia nigra and alpha-synuclein containing protein inclusions, called Lewy Bodies. The diagnosis of idiopathic PD is still based on the assessment of clinical criteria, leading to an insufficient diagnostic accuracy. Additionally, there is no biomarker available allowing the prediction of the disease course or monitoring the response to therapeutic approaches. So far, protein biomarker candidates such as alpha-synuclein have failed to improve diagnosis of PD. Circulating microRNAs (miRNAs) in body fluids are promising biomarker candidates for PD, as they are easily accessible by non- or minimally-invasive procedures and changes in their expression are associated with pathophysiological processes relevant for PD. Advances in miRNA analysis methods resulted in numerous recent publications on miRNAs as putative biomarkers. Here, we discuss the applicability of different body fluids as sources for miRNA biomarkers, highlight technical aspects of miRNA analysis and give an overview on published studies investigating circulating miRNAs as biomarker candidates for diagnosis of PD and other Parkinsonian syndromes.

Role of microRNAs in the main molecular pathways of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver malignant neoplasia. HCC is characterized by a poor prognosis. The need to find new molecular markers for its diagnosis and prognosis has led to a progressive increase in the number of scientific studies on this topic. MicroRNAs (miRNAs) are small non-coding RNA that play a role in almost all main cellular pathways. miRNAs are involved in the regulation of expression of the major tumor-related genes in carcinogenesis, acting as oncogenes or tumor suppressor genes. The aim of this review was to identify papers published in 2017 investigating the role of miRNAs in HCC tumorigenesis. miRNAs were classified according to their role in the main molecular pathways involved in HCC tumorigenesis: (1) mTOR; (2) Wnt; (3) JAK/STAT; (4) apoptosis; and (5) MAPK. The role of miRNAs in prognosis/response prediction was taken into consideration. Bearing in mind that the analysis of miRNAs in serum and other body fluids would be crucial for clinical management, the role of circulating miRNAs in HCC patients was also investigated. The most represented miRNA-regulated pathway in HCC is mTOR, but apoptosis, Wnt, JAK/STAT or MAPK pathways are also influenced by miRNA expression levels. These miRNAs could thus be used in clinical practice as diagnostic, prognostic or therapeutic targets for HCC treatment.

F10. DIFFERENTIAL EXPRESSION OF MICRORNAS IN CEREBROSPINAL FLUID AND PLASMA SAMPLES IN SCHIZOPHRENIA

BackgroundGenetic variants in miRNA genes and abnormalities in the concentration of microRNAs (miRNAs) in tissues and biological fluids have recently been associated with a diagnosis of schizophrenia. Most of these studies used post-mortem brain tissue or whole blood as the source of RNA. However, examination of microRNAs in cerebrospinal fluid (CSF) might provide an in vivo biomarker, more accurately reflecting expression level changes in the brain. To date, there are no studies that have investigated miRNA expression in CSF in patients with schizophrenia using small RNA-seq. In the past, our group had the opportunity of investigating the correlation between miRNA profiles in CSF and blood measured using microarray technology. Therefore, to expand our findings and use current cutting-edge technology, we measured miRNA profiles in CSF and plasma using small RNA-seq in a sample of patients with schizophrenia-spectrum disorder (SSD) diagnosis and healthy volunteers.MethodsTwenty-two SSD patients and 17 healthy volunteers underwent a lumbar puncture and a blood draw. 15–25 cc of CSF and 5–10 cc of peripheral blood were obtained from each subject. CSF and peripheral blood samples were centrifuged. CSF and plasma samples were aliquoted into 1 mL cryovials, and stored at -80C degrees. Vesicular RNA was extracted from 1 mL of CSF and plasma samples following the protocol from the Qiagen exoRNA easy kit. The BioScientific NextFlex RNA sequencing kit was used for library construction. Sequencing was done on HiSeq2500. Samples that had at least 50,000 reads going to mature miRNA sequences were included in the analysis. Differential expression analyses were conducted in R using the DESeq2 package in Bioconductor.ResultsIn the overall sample cohort, most subjects were male (66.7%), not Hispanic (81.0%) and black (48.7%). Mean age was 36.8 years (SD=12.3), There were no differences in age, sex, ethnicity or race between the patient and healthy control groups. In the patient group, 16 (72.7%) had schizophrenia, 5 (22.7%) had schizoaffective disorder and 1 (4.5%) had psychosis not otherwise specified. Differential expression (DE) analyses were conducted for 144 miRNAs in CSF and 354 miRNAs in plasma. After adjusting for multiple comparisons, DE analysis between patients and controls in CSF showed statistically significant higher levels in patients of miR-769-5p, miR-99b-3p, miR-107, miR-451a and miR-708-5. Similar analysis in plasma showed statistically significant higher levels in patients for miR-375, miR-204-5p, miR-942-5p, miR-6734-5p, miR-423-5p and miR-144-5p. Principal component analysis showed a clear separation between CSF and peripheral blood samples. Out of 443 miRNAs used to examine the relationship between CSF and plasma, 205 (46.3%) were detected in both plasma and CSF samples, 88 (19.9%) were detected only in CSF samples while 150 (33.9%) were detected only in plasma samples.DiscussionFive miRNAs were upregulated in CSF samples and six were upregulated in plasma samples of SSD patients compared to healthy volunteers. There was no overlap in the statistically significant upregulated miRNAs between CSF and plasma samples. Therefore, miRNA profiles in CSF and plasma have important quantitative and qualitative differences that may make them excellent, but different, candidate biofluids for biomarker discovery.

Serum miR-542-3p as a prognostic biomarker in osteosarcoma.

Emerging evidence has suggested that circulating microRNAs (miRNAs) in body fluids have novel diagnostic and prognostic significance for patients with malignant diseases. The lack of useful biomarkers is a crucial problem of osteosarcoma (OS); Previous study has reported that miR-542-3p was significantly upregulated in osteosarcoma tissues and miR-542-3p may be as an oncogene in osteosarcoma pathogenesis. In our study, we investigated the circulating miR-542-3p and its clinical relevance in osteosarcoma. Serum MiR-542-3p levels were determined by quantitative real-time PCR assays (qRT-PCR) in 76 patients with OS and 76 healthy volunteers. Patient survival analyses were performed by Kaplan-Meier analyses and Cox regression models. All statistical tests were two-sided. It was observed that the serum levels of miR-542-3p was significantly higher in patients with OS compared with the control groups (P< 0.01). High serum of miR-542-3p was significantly associated with advanced tumor stage and shorter survival (P< 0.01). ROC curve analysis calculated the ideal miR-542-3p cut-off value of 0.84 in prediction of OS, with a sensitivity of 53.8%, specificity of 93.6%, positive predictive value of 87.3% and negative predictive value of 63.7%. The results showed that serum miR-542-3p levels could serve as a non-invasive blood biomarker for tumor monitoring and prognostic prediction in osteosarcoma patients.

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR.

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This approach is appropriate for examining physiological changes in target gene expression levels. However, absolute quantification using better statistical analysis is preferable for a comprehensive assessment of gene expression levels. Absolute quantification is still not in common use. This report describes a protocol for measuring the absolute levels of plasma miRNA, using RT-qPCR with or without pre-amplification. A fixed volume (200 µL) of EDTA-plasma was prepared from the blood collected from the femoral vein of conscious cynomolgus monkeys (n = 50). Total RNA was extracted using commercially available system. Plasma miRNAs were quantified by probe-based RT-qPCR assays which contains miRNA-specific forward/reverse PCR primer and probe. Standard curves for absolute quantification were generated using commercially available synthetic RNA oligonucleotides. A synthetic cel-miR-238 was used as an external control for normalization and quality assessment. The miRNAs that showed quantification cycle (Cq) values above 35 were pre-amplified prior to the qPCR step. Among the 8 miRNAs examined, miR-122, miR-133a, and miR-192 were detectable without pre-amplification, whereas miR-1, miR-206, and miR-499a required pre-amplification because of their low expression levels. MiR-208a and miR-208b were not detectable even after pre-amplification. Sample processing efficiency was evaluated by the Cq values of the spiked cel-miR-238. In this assay method, technical variation was estimated to be less than 3-fold and the lower limit of quantification (LLOQ) was 102 copy/µL, for most of the examined miRNAs. This protocol provides a better estimate of the quantity of plasma miRNAs, and allows quality assessment of corresponding data from different studies. Considering the low number of miRNAs in body fluids, pre-amplification is useful to enhance detection of poorly expressed miRNAs.

MicroRNAs as biomarkers of kidney allograft injuries ischemia reperfusion and acute rejection

Renal transplantation provides a significant increase in life expectancy for patients with end-stage kidney diseases. Despite the unequivocal progresses made in the last decades, many grafts injuries still jeopardize graft function leading to its loss. Currently, the allograft biopsy is the gold standard for diagnosing such conditions. However, it has many limitations including risk and cost. Therefore, it is desirable to develop non-invasive biomarkers able to lead to the diagnosis of graft injuries. This is particularly true during the delayed graft function (DGF) period in which the functional parameters are not available. MicroRNAs regulate their target genes through mechanisms of translational repression and messenger RNA (mRNA) degradation. A single miRNA can regulate the expression of hundreds of mRNAs and proteins, deregulation of miRNAs can lead to disruption and suppression of genes that operate in intracellular signaling cascades leading to disease conditions. Technological advances have allowed the accurate detection of miRNAs in biological fluids providing either qualitative or quantitative results. Here we reviewed the literature in which miRNAs were analyzed as injury markers in renal transplantation mainly for contribute the diagnosis of acute rejection (AR). In cross sectional studies miRNAs were found to be useful markers of AR in renal transplant patients and their heightened expression by may became useful on aiding the diagnosis of AR during the DGF period.

Differential Expression of Serum MicroRNAs Supports CD4⁺ T Cell Differentiation into Th2/Th17 Cells in Severe Equine Asthma.

MicroRNAs (miRNAs) regulate post-transcriptional gene expression and may be exported from cells via exosomes or in partnership with RNA-binding proteins. MiRNAs in body fluids can act in a hormone-like manner and play important roles in disease initiation and progression. Hence, miRNAs are promising candidates as biomarkers. To identify serum miRNA biomarkers in the equine model of asthma we investigated small RNA derived from the serum of 34 control and 37 asthmatic horses. These samples were used for next generation sequencing, novel miRNA identification and differential miRNA expression analysis. We identified 11 significantly differentially expressed miRNAs between case and control horses: eca-miR-128, eca-miR-744, eca-miR-197, eca-miR-103, eca-miR-107a, eca-miR-30d, eca-miR-140-3p, eca-miR-7, eca-miR-361-3p, eca-miR-148b-3p and eca-miR-215. Pathway enrichment using experimentally validated target genes of the human homologous miRNAs showed a significant enrichment in the regulation of epithelial-to-mesenchymal transition (key player in airway remodeling in asthma) and the phosphatidylinositol (3,4,5)-triphosphate (PIP3) signaling pathway (modulator of CD4+ T cell maturation and function). Downregulated miR-128 and miR-744 supports a Th2/Th17 type immune response in severe equine asthma.

In Vitro Validation of miRNA-Mediated Gene Expression Linked to Drug Metabolism.

Pharmacogenomic approaches used to investigate how genes affect drug responses are critical for designing personalized therapies aimed at maximizing efficacy and minimizing adverse effects. Drug efficacy is often dependent on the sequence and expression levels of drug target genes or those involved in the metabolism and transport of the therapeutic agent. Expression of these genes, in turn, is negatively regulated by small noncoding miRNAs. The levels of miRNAs in bodily fluids have been studied extensively as potential diagnostic and prognostic biomarkers. Studies have shown that miRNAs regulate multiple genes and sequence homology is used to predict which genes are subject to regulation by a particular miRNA. Once a gene is identified as a potential target for an miRNA of interest, experiments are undertaken to confirm that the miRNA interacts with the target gene and can alter its level of expression and/or its activity. For example, the differential expression of miRNAs in whole blood obtained from good and poor responders to ketamine has been reported both prior to, and following treatment for complex regional pain syndrome. In this case, hsa-miR-548d-5p was significantly lower in poor responders relative to good responders. This miRNA was predicted to target UDP-glucuronyl transferase 1A1 (UGT1A1), a key drug metabolizing enzyme. Described in this unit are protocols used to confirm miR-548d-5p-mediated UGT1A1 regulation. The approaches described can be employed broadly for the validation of miRNA-mediated negative regulation of any gene. Determining miRNA-mediated regulation of enzymes and transporters affecting drug metabolism is a critical step in designing personalized therapy and for understanding the mechanisms responsible for variations in the responses to therapeutic agents. © 2017 by John Wiley & Sons, Inc.

Circulating MicroRNA-92b-3p as a Novel Biomarker for Monitoring of Synovial Sarcoma

The lack of useful biomarkers is a crucial problem for patients with soft tissue sarcomas (STSs). Emerging evidence has suggested that circulating microRNAs (miRNAs) in body fluids have novel impact as biomarkers for patients with malignant diseases, but their significance in synovial sarcoma (SS) patients remains unknown. Initial global miRNA screening using SS patient serum and SS cell culture media identified a signature of four upregulated miRNAs. Among these candidates, miR-92b-3p secretion from SS cells was confirmed, which was embedded within tumour-derived exosomes rather than argonaute-2. Animal experiments revealed a close correlation between serum miR-92b-3p levels and tumour dynamics. Clinical relevance was validated in two independent clinical cohorts, and we subsequently identified that serum miR-92b-3p levels were significantly higher in SS patients in comparison to that in healthy individuals. Moreover, serum miR-92b-3p was robust in discriminating patients with SS from the other STS patients and reflected tumour burden in SS patients. Overall, liquid biopsy using serum miR-92b-3p expression levels may represent a novel approach for monitoring tumour dynamics of SS.

Asymmetric RNA Distribution among Cells and Their Secreted Exosomes: Biomedical Meaning and Considerations on Diagnostic Applications.

Over the past few years, exosomes and their RNA cargo have been extensively studied because of the fascinating biological roles they play in cell-to-cell communication, including the signal exchange among cancer, stromal, and immune cells, leading to modifications of tumor microenvironment. RNAs, especially miRNAs, stored within exosomes, seem to be among the main determinants of such signaling: their sorting into exosomes appears to be cell-specific and related to cellular physiopathology. Accordingly, the identification of exosomal miRNAs in body fluids from pathological patients has become one of the most promising activity in the field of biomarker discovery. Several analyses on the qualitative and quantitative distribution of RNAs between cells and their secreted exosomes have given rise to questions on whether and how accurately exosomal RNAs would represent the transcriptomic snapshot of the physiological and pathological status of secreting cells. Although the exact molecular mechanisms of sorting remain quite elusive, many papers have reported an evident asymmetric quantitative distribution of RNAs between source cells and their exosomes. This phenomenon could depend both on passive and active sorting mechanisms related to: (a) RNA turnover; (b) maintaining the cytoplasmic miRNA:target equilibrium; (c) removal of RNAs not critical or even detrimental for normal or diseased cells. These observations represent very critical issues in the exploitation of exosomal miRNAs as cancer biomarkers. In this review, we will discuss how much the exosomal and corresponding donor cell transcriptomes match each other, to better understand the actual reliability of exosomal RNA molecules as pathological biomarkers reflecting a diseased status of the cells.

Ultrasensitive Electrochemical Detection of miRNA-21 Using a Zincfinger Protein Specific to DNA−RNA Hybrids and Redox Cycling

Both high sensitivity and high specificity are crucial for detection of miRNAs that have emerged as important clinical biomarkers. Just Another Zinc finger proteins (JAZ, ZNF346) bind preferably (but nonsequence-specifically) to DNA−RNA hybrids over single-stranded RNAs, single-stranded DNAs, and double-stranded DNAs. We present an ultrasensitive and highly specific electrochemical method for miRNA-21 detection based on the selective binding of JAZ to the DNA−RNA hybrid formed between a DNA capture probe and a target miRNA-21. This enables us to use chemically stable DNA as a capture probe instead of RNA as well as to apply a standard sandwich-type assay format to miRNA detection. High signal amplification is obtained by (i) enzymatic amplification by alkaline phosphatase (ALP) coupled with (ii) electrochemical-chemical-chemical (ECC) redox cycling involving an ALP product (hydroquinone). Low nonspecific adsorption of ALP-conjugated JAZ is obtained using a polymeric self-assembled-monolayer-modified and casein-treated indium−tin oxide electrode. The detection method can discriminate between target miRNA-21 and nontarget nucleic acids (DNA−DNA hybrid, single-stranded DNA, miRNA-125b, miRNA-155, single-base mismatched miRNA, and three-base mismatched miRNA). The detection limits for miRNA-21 in buffer and 10-fold diluted serum are approximately 2 and 30 fM, respectively, dicating that the detection method is ultrasensitive. This detection method can be readily extended to multiplex detection of miRNAs with only one ALP-conjugated JAZ probe due to its nonsequence-specific binding character. We also believe that the method could offer a promising solution for point-of-care testing of miRNAs in body fluids.

Identification of reference miRNAs in plasma useful for the study of oestrogen-responsive miRNAs associated with acquired Protein S deficiency in pregnancy

BackgroundAccumulating evidence indicate that circulating microRNAs (miRNAs) are useful independent non-invasive biomarkers, with unique miRNA signatures defined for various pathophysiological conditions. However, there are no established universal housekeeping miRNAs for the normalisation of miRNAs in body fluids. We have previously identified an oestrogen-responsive miRNA, miR-494, in regulating the anticoagulant, Protein S, in HuH-7 liver cells. Moreover, increased thrombotic risk associated with elevated circulating oestrogen levels is frequently observed in pregnant women and oral contraceptive users. In order to identify other oestrogen-responsive miRNAs, including miR-494, that may be indicative of increased thrombotic risk in plasma, we used nanoString analysis to identify robust and stable endogenous reference miRNAs for the study of oestrogen-responsive miRNAs in plasma.ResultsWe compared the plasma miRNA expression profile of individuals with: (1) Low circulating oestrogens (healthy men and non-pregnant women not taking oral contraceptives), (2) High circulating synthetic oestrogens, (women taking oral contraceptives) and (3) High circulating natural oestrogens (pregnant females >14 weeks gestation). From the nanoString analyses, 11 candidate reference miRNAs which exhibited high counts and not significantly differentially expressed between groups were selected for validation using realtime quantitative polymerase chain reaction (RT-qPCR) and digital droplet PCR (DDPCR) in pooled plasma samples, and the stability of their expression evaluated using NormFinder and BestKeeper algorithms. Four miRNAs (miR-25-5p, miR-188-5p, miR-222-3p and miR-520f) demonstrated detectable stable expression between groups and were further analysed by RT-qPCR in individual plasma samples, where miR-188-5p and miR-222-3p expression were identified as a stable pair of reference genes. The miRNA reference panel consisting of synthetic spike-ins cel-miR-39 and ath-miR159a, and reference miRNAs, miR-188-5p and miR-222-3p was useful in evaluating fold-change of the pregnancy-associated miRNA, miR-141-3p, between groups.ConclusionThe miRNA reference panel will be useful for normalising qPCR data comparing miRNA expression between men and women, non-pregnant and pregnant females, and the potential effects of endogenous and synthetic oestrogens on plasma miRNA expression.