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MicroRNAs (miRNAs) play essential roles in post-transcriptional gene expression and are also found freely circulating in bodily fluids such as blood. Dysregulated miRNA signatures have been associated with many diseases including cancer, and miRNA profiling from liquid biopsies offers a promising strategy for cancer diagnosis, prognosis and monitoring. Here, we develop size-encoded molecular probes that can be used for simultaneous electro-optical nanopore sensing of miRNAs, allowing for ultrasensitive, sequence-specific and multiplexed detection directly in unprocessed human serum, in sample volumes as small as 0.1 μl. We show that this approach allows for femtomolar sensitivity and single-base mismatch selectivity. We demonstrate the ability to simultaneously monitor miRNAs (miR-141-3p and miR-375-3p) from prostate cancer patients with active disease and in remission. This technology can pave the way for next generation of minimally invasive diagnostic and companion diagnostic tests for cancer. miRNA profiling from patient blood can be used for cancer diagnosis. Here the authors present an electro-optical nanopore sensing platform which allows sensitive and specific miRNA detection directly in human serum and apply to monitoring of miR-141-3p and miR-375-3p in different stage of prostate cancer.

Chronic sleep disruption and shift work elevate the risk of neurodegeneration and Alzheimer's disease (AD). While disrupted sleep affects canonical AD biomarkers, its impact on other mechanisms, such as circulating microRNAs (miRNAs), remains less understood. Therefore, we here examined the effects of overnight wakefulness on plasma levels of several miRNAs implicated in neurodegeneration and AD, as well as in sleep and circadian regulation—namely miR‐127‐3p, miR‐132‐3p, and miR‐142‐3p. Following a baseline period in each highly controlled in‐lab session, in total 15 healthy normal‐weight young men underwent two conditions on separate occasions, in randomised order: a night of normal sleep, and a night of sustained wakefulness. After overnight wakefulness, morning plasma levels of miR‐127‐3p and miR‐142‐3p were significantly elevated compared with post‐sleep levels. These changes were not associated with the significant increase in self‐reported morning stress levels observed after wakefulness compared with sleep. This study is the first to demonstrate that a single night of wakefulness—mimicking overnight shift work—significantly elevates circulating levels of miR‐127‐3p and miR‐142‐3p in humans. These findings, though based on a limited sample size, suggest a potential molecular link between sleep loss and neurodegeneration, warranting further investigation. Trial Registration: Clinical Trial number: NCT01800253; www.clinicaltrials.gov

Obesity is a complex condition that is characterized by excessive fat accumulation, which can lead to the development of metabolic disorders, such as type 2 diabetes mellitus, nonalcoholic fatty liver disease and cardiovascular diseases. Evidence is accumulating that circulating microRNAs (miRNAs) act as a new class of endocrine factor. These miRNAs are released by many types of tissue, including adipose tissues. miRNAs might serve as endocrine and paracrine messengers that facilitate communication between donor cells and tissues with receptor cells or target tissues, thereby potentially having important roles in metabolic organ crosstalk. Moreover, many miRNAs are closely associated with the differentiation of adipocytes and are dysregulated in obesity. As such, circulating miRNAs are attractive potential biomarkers and hold promise for the development of miRNA-based therapeutics (such as miRNA mimetics, anti-miRNA oligonucleotides and exosomes loaded with miRNA) for obesity and related disorders. Here we review the latest research progress on the roles of circulating miRNAs in metabolic organ crosstalk. In addition, we discuss the clinical potential of circulating miRNAs as feasible biomarkers for the assessment of future risk of metabolic disorders and as therapeutic targets in obesity and related diseases.

Background Circulating microRNAs have shown promise as non-invasive biomarkers and predictors of disease activity. Prior asthma studies using clinical, biochemical and genomic data have not shown excellent prediction of exacerbation. We hypothesized that a panel of circulating microRNAs in a pediatric asthma cohort combined with an exacerbation clinical score might predict exacerbation better than the latter alone. Methods Serum samples from 153 children at randomization in the Childhood Asthma Management Program were profiled for 754 microRNAs. Data dichotomized for asthma exacerbation one year after randomization to inhaled corticosteroid treatment were used for binary logistic regression with miRNA expressions and exacerbation clinical score. Results 12 of 125 well-detected circulating microRNAs had significant odd ratios for exacerbation with miR-206 being most significant. Each doubling of expression of the 12 microRNA corresponded to a 25–67% increase in exacerbation risk. Stepwise logistic regression yielded a 3-microRNA model (miR-146b, miR-206 and miR-720) that, combined with the exacerbation clinical score, had excellent predictive power with a 0.81 AUROC. These 3 microRNAs were involved in NF-kβ and GSK3/AKT pathways. Conclusions This combined circulating microRNA-clinical score model predicted exacerbation in asthmatic subjects on inhaled corticosteroids better than each constituent feature alone. Trial registration ClinicalTrials.gov Identifier: NCT00000575 .

Abstract Context Obesity is a global epidemic and an independent risk factor for several diseases. miRNAs are gaining interest as early molecular regulators of various pathological processes. Objective To examine the miRNA signatures in women who are obese and determine the response of miRNAs to acute weight loss. Methods Plasma samples were collected from women who are obese (n = 80) before and after acute weight loss (mean, 7.2%). Plasma samples from age-matched lean volunteers (n = 80) were used as controls. Total RNA was extracted from the plasma samples and subjected to NanoString analysis of 822 miRNAs. The expression level of candidate miRNAs was validated in all participants using quantitative real-time PCR analysis. Results NanoString analysis identified substantial dysregulation of 21 miRNAs in women who are obese that were associated with impaired glucose tolerance, senescence, cardiac hypertrophy, angiogenesis, inflammation, and cell death. Acute weight loss reversed the expression pattern of 18 of these miRNAs toward those seen in the lean control group. Furthermore, real-time PCR validation of all the samples for 13 miRNAs with at least twofold upregulation or downregulation confirmed substantial dysregulation of all the chosen miRNAs in women who are obese at baseline. After acute weight loss, the levels of seven miRNAs in women who are obese and who are lean were comparable, with no statistically significant evidence for differences between the two groups. Conclusions Our study has provided evidence that the circulating miRNAs associated with various disorders are dysregulated in women who are obese. We also found that seven of these miRNAs showed levels comparable to those in lean controls after acute weight loss in women who are obese. Acute weight loss normalized dysregulated circulating microRNAs in women who are obese, providing evidence for microRNAs as potential biomarkers to determine the beneficial effects of weight loss.

Primary prevention of premature death is a public health concern worldwide. Circulating microRNAs (miRNAs) have been described as potential diagnostic biomarkers for diseases as cancer and cardiovascular disease (CVD). This case-cohort study aimed to investigate the potential relationship between circulating miRNAs and the risk of premature death. A total of 39,242 subjects provided baseline serum samples in 1988–1990. Of these, 345 subjects who died of intrinsic disease (< 65 years old) and for which measurable samples were available were included in this study. We randomly selected a sub-cohort of 879 subjects. Circulatring miR-21, miR-29a, and miR-126 were determined using qRT-PCR. Conditional logistic regression models were used to analyse the data with respect to stratified miRNA levels. Multivariable logistic regression revealed that subjects with high circulating miR-21 and miR-29a individual levels had a significantly higher risk of total death, cancer death, and CVD death than those with medium miR-21 and miR-29a individual levels. Conversely, subjects with low circulating miR-126 levels had a significantly higher risk of total death than those with medium levels. This suggests that circulating miRNAs are associated with the risk of premature death from cancer and CVD, identifying them as potential biomarkers for early detection of high-risk individuals.

Sample collection, processing, storage and isolation methods constitute pre-analytic factors that can influence the quality of samples used in research and clinical practice. With regard to biobanking practices, a critical point in the sample's life chain is storage, particularly long-term storage. Since most studies examine the influence of different temperatures (4°C, room temperature) or delays in sample processing on sample quality, there is only little information on the effects of long-term storage at ultra-low (vapor phase of liquid nitrogen) temperatures on biomarker levels. Among these biomarkers, circulating miRNAs hold great potential for diagnosis or prognosis for a variety of diseases, like cancer, infections and chronic diseases, and are thus of high interest in several scientific questions. We therefore investigated the influence of long-term storage on levels of eight circulating miRNAs (miR-103a-3p, miR-191-5p, miR-124-3p, miR-30c-5p, miR-451a, miR-23a-3p, miR-93-5p, miR-24-3p, and miR-33b-5p) from 10 participants from the population-based cohort study KORA. Sample collection took place during the baseline survey S4 and the follow-up surveys F4 and FF4, over a time period spanning from 1999 to 2014. The influence of freeze-thaw (f/t) cycles on miRNA stability was also investigated using samples from volunteers (n = 6). Obtained plasma samples were profiled using Exiqon's miRCURYTM real-time PCR profiling system, and repeated measures ANOVA was used to check for storage or f/t effects. Our results show that detected levels of most of the studied miRNAs showed no statistically significant changes due to storage at ultra-low temperatures for up to 17 years; miR-451a levels were altered due to contamination during sampling. Freeze-thawing of one to four cycles showed an effect only on miR-30c-5p. Our results highlight the robustness of this set of circulating miRNAs for decades of storage at ultra-low temperatures and several freeze-thaw cycles, which makes our findings increasingly relevant for research conducted with biobanked samples.

MicroRNAs (miRNAs) are a class of small regulatory RNAs around 21-25 nucleotides in length which govern many aspects of immunity including the host innate and adaptive responses to infection. RT-qPCR studies of select microRNAs show that vaccination alters the expression circulating microRNAs but the effect of vaccination on the global microRNA population (i.e. micronome) has never been studied. To describe vaccine associated changes in the expression of microRNAs 21 days after vaccination in children receiving a pandemic influenza (H1N1) vaccination. Serum samples were obtained from children aged 6 months to 12 years enrolled in an open label randomised control trial of two pandemic influenza (H1N1) vaccines, in which participants received either ASO3B adjuvanted split virion or a whole virion non-adjuvanted vaccine. MicroRNA expression was profiled in a discovery cohort of participants prior to, and 21 days after vaccination using an Agilent microarray platform. Findings were followed up by RT-qPCR in the original discovery cohort and then in a validation cohort of participants taken from the same study. 44 samples from 22 children were assayed in a discovery cohort. The microarray results revealed 19 microRNAs were differentially expressed after vaccination after adjustment for multiple testing. The microarray detected ubiquitous expression of several microRNAs which could not be validated by RT-qPCR, many of which have little evidence of existence in publicly available RNA sequencing data. Real time PCR (RT-qPCR) confirmed downregulation of miR-142-3p in the discovery cohort. These findings were not replicated in the subsequent validation cohort (n = 22). This study is the first study to profile microRNA expression after vaccination. An important feature of this study is many of the differentially expressed microRNAs could not be detected and validated by RT-qPCR. This study highlights the care that should be taken when interpreting omics biomarker discovery, highlighting the need for supplementary methods to validate microRNA microarray findings, and emphasises the importance of validation cohorts. Data from similar studies which do not meet these requirements should be interpreted with caution.

Night shift work increases risk of metabolic disorders, particularly obesity and insulin resistance. While the underlying mechanisms are unknown, evidence points to misalignment of peripheral oscillators causing metabolic disturbances. A pathway conveying such misalignment may involve exosome-based intercellular communication. Fourteen volunteers were assigned to a simulated day shift (DS) or night shift (NS) condition. After 3 days on the simulated shift schedule, blood samples were collected during a 24-h constant routine protocol. Exosomes were isolated from the plasma samples from each of the blood draws. Exosomes were added to naïve differentiated adipocytes, and insulin-induced pAkt/Akt expression changes were assessed. ChIP-Seq analyses for BMAL1 protein, mRNA microarrays and exosomal miRNA arrays combined with bioinformatics and functional effects of agomirs and antagomirs targeting miRNAs in NS and DS exosomal cargo were examined. Human adipocytes treated with exosomes from the NS condition showed altered Akt phosphorylation responses to insulin in comparison to those treated with exosomes from the DS condition. BMAL1 ChIP-Seq of exosome-treated adipocytes showed 42,037 binding sites in the DS condition and 5538 sites in the NS condition, with a large proportion of BMAL1 targets including genes encoding for metabolic regulators. A significant and restricted miRNA exosomal signature emerged after exposure to the NS condition. Among the exosomal miRNAs regulated differentially after 3 days of simulated NS versus DS, proof-of-concept validation of circadian misalignment signaling was demonstrated with hsa-mir-3614-5p. Exosomes from the NS condition markedly altered expression of key genes related to circadian rhythm in several cultured cell types, including adipocytes, myocytes, and hepatocytes, along with significant changes in 29 genes and downstream gene network interactions. Our results indicate that a simulated NS schedule leads to changes in exosomal cargo in the circulation. These changes promote reduction of insulin sensitivity of adipocytes in vitro and alter the expression of core clock genes in peripheral tissues. Circulating exosomal miRNAs may play an important role in metabolic dysfunction in NS workers by serving as messengers of circadian misalignment to peripheral tissues.

The plasma levels of tissue-specific microRNAs can be used as diagnostic, disease severity and prognostic biomarkers for chronic and acute diseases and drug-induced injury. Thereby, the combination of diverse microRNAs into biomarker signatures using multivariate statistics seems especially powerful from the perspective of tissue and condition specific microRNA shedding into the plasma. Although next-generation sequencing (NGS) technology enables one to analyse circulating microRNAs on a genome-scale level, it suffers from potential biases (e.g., adapter ligation bias) and lacks absolute transcript quantitation as well as tailor-made quality controls. In order to develop a robust NGS discovery assay for genome-scale quantitation of circulating microRNAs, we first evaluated the sensitivity, repeatability and ligation bias of four commercially available small RNA library preparation protocols. The protocol from RealSeq Biosciences was selected based on its performance and usability and coupled with a novel panel of exogenous small RNA spike-in controls to enable quality control and absolute quantitation, thus ensuring comparability of data across independent NGS experiments. The established microRNA Next-Generation-Sequencing Discovery Assay (miND) was validated for its relative accuracy, precision, analytical measurement range and sequencing bias and was considered fit-for-purpose for microRNA biomarker discovery. Summarized, all these criteria were met, and thus, our analytical platform is considered fit-for-purpose for microRNA biomarker discovery from biofluids in the setting of any diagnostic, prognostic or patient stratification need. The established miND assay was tested on serum, cerebrospinal fluid (CSF), synovial fluid (SF) and extracellular vesicles (EV) extracted from cell culture medium of primary cells and proved its potential to be used across different sample types.