Associations between circulating microRNAs and lipid-rich coronary plaques measured with near-infrared spectroscopy.

Lipid-rich coronary atherosclerotic plaques often cause myocardial infarction (MI), and circulating biomarkers that reflect lipid content may predict risk of MI. We investigated the association between circulating microRNAs (miRs) are lipid-rich coronary plaques in 47 statin-treated patients (44 males) with stable coronary artery disease undergoing percutaneous coronary intervention. We assessed lipid content in non-culprit coronary artery lesions with near-infrared spectroscopy and selected the 4 mm segment with the highest measured lipid core burden index (maxLCBI4mm). Lipid-rich plaques were predefined as a lesion with maxLCBI4mm ≥ 324.7. We analyzed 177 circulating miRs with quantitative polymerase chain reaction in plasma samples. The associations between miRs and lipid-rich plaques were analyzed with elastic net. miR-133b was the miR most strongly associated with lipid-rich coronary plaques, with an estimated 18% increase in odds of lipid-rich plaques per unit increase in miR-133b. Assessing the uncertainty by bootstrapping, miR-133b was present in 82.6% of the resampled dataset. Inclusion of established cardiovascular risk factors did not attenuate the association. No evidence was found for an association between the other analyzed miRs and lipid-rich coronary plaques. Even though the evidence for an association was modest, miR-133b could be a potential biomarker of vulnerable coronary plaques and risk of future MI. However, the prognostic value and clinical relevance of miR-133b needs to be assessed in larger cohorts.

An Epidemiological Systematic Review with Meta-Analysis on Biomarker Role of Circulating MicroRNAs in Breast Cancer Incidence.

Breast cancer (BC) is a multifactorial disease caused by an interaction between genetic predisposition and environmental exposures. MicroRNAs are a group of small non-coding RNA molecules, which seem to have a role either as tumor suppressor genes or oncogenes and seem to be related to cancer risk factors. We conducted a systematic review and meta-analysis to identify circulating microRNAs related to BC diagnosis, paying special attention to methodological problems in this research field. A meta-analysis was performed for microRNAs analyzed in at least three independent studies where sufficient data to make analysis were presented. Seventy-five studies were included in the systematic review. A meta-analysis was performed for microRNAs analyzed in at least three independent studies where sufficient data to make analysis were presented. Seven studies were included in the MIR21 and MIR155 meta-analysis, while four studies were included in the MIR10b metanalysis. The pooled sensitivity and specificity of MIR21 for BC diagnosis were 0.86 (95%CI 0.76-0.93) and 0.84 (95%CI 0.71-0.92), 0.83 (95%CI 0.72-0.91) and 0.90 (95%CI 0.69-0.97) for MIR155, and 0.56 (95%CI 0.32-0.71) and 0.95 (95%CI 0.88-0.98) for MIR10b, respectively. Several other microRNAs were found to be dysregulated, distinguishing BC patients from healthy controls. However, there was little consistency between included studies, making it difficult to identify specific microRNAs useful for diagnosis.

Circulating microRNAs as putative mediators in the association between short-term exposure to ambient air pollution and cardiovascular biomarkers.

BACKGROUND: Exposure to ambient air pollution is associated with increased cardiovascular morbidity and mortality. Circulating microRNAs (miRNAs) may mediate cardiovascular effects of exposure to air pollution. This study aims to investigate whether circulating miRNAs mediate the associations between short-term human exposure to ambient air pollution and cardiovascular biomarkers. METHODS: Twenty-four healthy adults residing in the Research Triangle area of North Carolina, USA were enrolled between December 2016 and July 2019. Circulating miRNAs, protein, and lipid biomarkers were assessed repeatedly for 3 sessions separated by at least 7 days. Linear mixed-effects models were used to assess the associations between air pollutant concentrations obtained from nearby air quality monitoring stations and miRNAs controlling for covariates including omega-3 index, relative humidity, and temperature. miRNAs that were significantly altered were then matched with protein or blood lipid biomarkers using either Ingenuity Pathway Analysis or a literature search. A mediation analysis was performed to test the statistical significance of miRNA's mediating effects between exposure to air pollution and cardiovascular biomarkers. RESULTS: Short-term exposure to ambient fine particulate matter (PM2.5), ozone (O3), and nitrogen dioxide (NO2) was associated with changes in 11, 9, and 24 circulating miRNAs, respectively. Pathway analysis showed that several miRNAs including miR-125b-5p, miR-144-5p, miR-26a-5p, and miR-34a-5p may mediate the effects of air pollutant exposure on the changes of downstream protein / lipid biomarkers including serum amyloid A (SAA), C-reactive protein (CRP), soluble vascular adhesive molecules 1 (sICAM1), total cholesterol, and high-density lipoproteins (HDL). Mediation analysis showed that only miR-26a-5p significantly mediated air pollutant (PM2.5 and NO2)-induced effects on blood CRP and total cholesterol levels. For example, 34.1% of PM2.5-associated changes in CRP were significantly mediated by miR-26a-5p at lag4 [indirect effects, 0.06 (0.02, 0.10), P = 0.005]. Similarly, the proportions of indirect effects of miR-26a-5p on the association between NO2 exposure and CRP were 46.8% at lag2 [0.06 (0.02, 0.11), P = 0.003], 61.2% at lag3 [0.05 (0.00, 0.09), P = 0.04], and 30.8% at 5-day moving average [0.06 (0.02, 0.10), P = 0.01]. In addition, omega-3 index may be a significant modifying factor of the mediated effects of miRNAs. CONCLUSIONS: This study demonstrates that short-term exposure to ambient PM2.5, O3, and NO2 was associated with specific circulating miRNAs, and some of which may mediate their effects on the downstream inflammation and blood lipid markers.

A MicroRNA Next-Generation-Sequencing Discovery Assay (miND) for Genome-Scale Analysis and Absolute Quantitation of Circulating MicroRNA Biomarkers.

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.

A Systematic Review of Circulatory microRNAs in Major Depressive Disorder: Potential Biomarkers for Disease Prognosis.

Major depressive disorder (MDD) is a neuropsychiatric disorder, which remains challenging to diagnose and manage due to its complex endophenotype. In this aspect, circulatory microRNAs (cimiRNAs) offer great potential as biomarkers and may provide new insights for MDD diagnosis. Therefore, we systemically reviewed the literature to explore various cimiRNAs contributing to MDD diagnosis and underlying molecular pathways. A comprehensive literature survey was conducted, employing four databases from 2012 to January 2021. Out of 1004 records, 157 reports were accessed for eligibility criteria, and 32 reports meeting our inclusion criteria were considered for in-silico analysis. This study identified 99 dysregulated cimiRNAs in MDD patients, out of which 20 cimiRNAs found in multiple reports were selected for in-silico analysis. KEGG pathway analysis indicated activation of ALS, MAPK, p53, and P13K-Akt signaling pathways, while gene ontology analysis demonstrated that most protein targets were associated with transcription. In addition, chromosomal location analysis showed clustering of dysregulated cimiRNAs at proximity 3p22-p21, 9q22.32, and 17q11.2, proposing their coregulation with specific transcription factors primarily involved in MDD physiology. Further analysis of transcription factor sites revealed the existence of HIF-1, REST, and TAL1 in most cimiRNAs. These transcription factors are proposed to target genes linked with MDD, hypothesizing that first-wave cimiRNA dysregulation may trigger the second wave of transcription-wide changes, altering the protein expressions of MDD-affected cells. Overall, this systematic review presented a list of dysregulated cimiRNAs in MDD, notably miR-24-3p, let 7a-5p, miR-26a-5p, miR135a, miR-425-3p, miR-132, miR-124 and miR-16-5p as the most prominent cimiRNAs. However, various constraints did not permit us to make firm conclusions on the clinical significance of these cimiRNAs, suggesting the need for more research on single blood compartment to identify the biomarker potential of consistently dysregulated cimiRNAs in MDD, as well as the therapeutic implications of these in-silico insights.

MicroRNA Expression Profiling in Porcine Liver, Jejunum and Serum upon Dietary DON Exposure Reveals Candidate Toxicity Biomarkers.

Deoxynivalenol (DON), a frequent mycotoxin worldwide, impairs human and animal health. The response of microRNAs, small non-coding RNAs, to DON has been scarcely investigated, but holds remarkable potential for biomarker applications. Hence, we aimed to investigate DON-induced changes in the microRNA expression in porcine liver, jejunum and serum by combining targeted and untargeted analyses. Piglets received uncontaminated feed or feed containing 900 µg/kg and 2500 µg/kg DON for four weeks, followed by a wash-out period. In tissue, only slight changes in microRNA expression were detected, with ssc-miR-10b being downregulated in liver of DON-exposed piglets. In serum, several microRNAs were differentially expressed upon DON exposure, four of which were validated by qPCR (ssc-miR-16, ssc-miR-128, ssc-miR-451, ssc-miR-205). The serum microRNA response to DON increased over time and declined after removal of contaminated diets. Receiver operating curve analyses for individual microRNAs were significant, and a combination of the four microRNAs increased the predictive capacity for DON exposure. Predicted microRNA target genes showed enrichment of several pathways including PIK3-AKT, Wnt/ß-catenin, and adherens junctions. This study gives, for the first time, a comprehensive view of the porcine microRNA response to DON, providing a basis for future research on microRNAs as biomarkers for mycotoxins.

Biosensors, microfluidics systems and lateral flow assays for circulating microRNA detection: A review.

MicroRNAs (miRNAs) are short RNA sequences found in eukaryotic cells and they are involved in several diseases pathogenesis including different types of cancers, metabolic and cardiovascular disorders. Thus, miRNAs circulating in serum, plasma, and other body fluids are employed as biomarkers for diagnostic and prognostic purposes and in assessment of drug response. Thus, various methods have been developed for detection of miRNAs including northern blotting, reverse transcriptase polymerase chain reaction (RT-PCR), next-generation sequencing, microarray, and isothermal amplification that are recognized as traditional methods. Considering the importance of early diagnosis and treatment of miRNAs-related diseases, development of simple, one-step, sensitive methods is of great interest. Nowadays developing technologies including lateral flow assay, biosensors (optical and electrochemical) and microfluidic systems which are simple fast responding, user-friendly, and are enabled with visible detection have gained considerable attention. This review briefly discusses miRNAs detection' methods, with a particular focus on lateral flow assay, biosensors, and microfluidic systems as novel and practical procedures.

Circulating microRNAs Signature for Predicting Response to GLP1-RA Therapy in Type 2 Diabetic Patients: A Pilot Study.

Type 2 diabetes (T2D) represents one of the major health issues of this century. Despite the availability of an increasing number of anti-hyperglycemic drugs, a significant proportion of patients are inadequately controlled, thus highlighting the need for novel biomarkers to guide treatment selection. MicroRNAs (miRNAs) are small non-coding RNAs, proposed as useful diagnostic/prognostic markers. The aim of our study was to identify a miRNA signature occurring in responders to glucagon-like peptide 1 receptor agonists (GLP1-RA) therapy. We investigated the expression profile of eight T2D-associated circulating miRNAs in 26 prospectively evaluated diabetic patients in whom GLP1-RA was added to metformin. As expected, GLP1-RA treatment induced significant reductions of HbA1c and body weight, both after 6 and 12 months of therapy. Of note, baseline expression levels of the selected miRNAs revealed two distinct patient clusters: "high expressing" and "low expressing". Interestingly, a significantly higher percentage of patients in the high expression group reached the glycemic target after 12 months of treatment. Our findings suggest that the evaluation of miRNA expression could be used to predict the likelihood of an early treatment response to GLP1-RA and to select patients in whom to start such treatment, paving the way to a personalized medicine approach.

Assessment of the Diagnostic Efficiency of a Liquid Biopsy Assay for Early Detection of Gastric Cancer.

Importance: Noninvasive detection of early-stage disease is a key strategy for reducing gastric cancer (GC)-associated patient mortality. Objective: To establish a novel, noninvasive, microRNA (miRNA)-based signature for the early detection of GC using a comprehensive biomarker discovery approach with retrospective and prospective validation. Design, Setting, and Participants: This diagnostic study was conducted in 4 phases using publicly available genome sequences and tissue samples from patients at an academic medical center in Japan, and validated with retrospective multicenter cohorts of patients with GC. Three tissue miRNA data sets were used to identify a miRNA signature that discriminated GC vs normal tissues. The robustness of this signature was assessed in serum from 2 retrospective cohorts of patients with GC. A risk-scoring model was derived, then the performance of the miRNA signature was evaluated in a prospective cohort of patients with GC. The robustness of the miRNA signature was compared with current blood-based markers, and a cost-effectiveness analysis of the miRNA signature against the current practice of endoscopy was performed. All clinical samples used for this study were collected and data analyzed between April 1997 and March 2018. Main Outcomes and Measures: Assessment of diagnostic efficiency on the basis of area under the curve (AUC), specificity, and sensitivity. Results: The data sets for the genome-wide expression profiling analysis stage included 598 total patient samples (284 [55.4%] from men; mean [SE] patient age, 65.7 [0.5] years). The resulting 10-miRNA signature was validated in 2 retrospective GC serum cohorts (586 patients; 348 [59.4%] men, mean [SE] age, 66.0 [0.7] years), which led to the establishment of a 5-miRNA signature (AUC, 0.90; 95% CI, 0.85-0.94) that also exhibited high levels of diagnostic performance in patients with stage I disease (AUC, 0.89; 95% CI, 0.83-0.94). A risk-scoring model was derived and the assay was optimized to a minimal number of miRNAs. The performance of the resulting 3-miRNA signature was then validated in a prospective cohort of patients with GC (349 patients; 124 [70.5%] men, median [range] age, 66.0 [0.66] years). The final 3-miRNA signature (miR-18a, miR-181b, and miR-335) exhibited high diagnostic accuracy in all stages of patients (AUC, 0.86; 95% CI 0.83-0.90), including in patients with stage I disease (AUC, 0.85; 95% CI, 0.79-0.91). Furthermore, this miRNA signature was superior to currently used blood markers and outperformed the endoscopic screening in a cost-effectiveness analysis (incremental cost-effectiveness ratio, CNY ¥16162.5 per quality-adjusted life-year [USD $2304.80 per quality-adjusted life-year]). Conclusions and Relevance: These results suggest the potential clinical significance of the 3-miRNA signature as a noninvasive, cost-effective, and facile assay for the early detection of GC.

Circulating skeletal muscle related microRNAs profile in Piedmontese cattle during different age.

Piedmontese cattle is known for double-muscle phenotype. MicroRNAs (miRNAs) play important role as regulators in skeletal muscle physiological processes, and we hypothesize that plasma miRNAs expression profiles could be affected by skeletal muscle growth status related to age. Plasma samples of cattle were collected during four different ages from first week of life until the time of commercial end of the fattening period before slaughter. Small-RNA sequencing data analysis revealed the presence of 40% of muscle-related miRNAs among the top 25 highly expressed miRNAs and, 19 miRNAs showed differential expression too. Using qRT-PCR, we validated in a larger bovine population, miRNAs involved in skeletal muscle physiology pathways. Comparing new-born with the other age groups, miR-10b, miR-126-5p, miR-143 and miR-146b were significantly up-regulated, whereas miR-21-5p, miR-221, miR-223 and miR-30b-5p were significantly down-regulated. High expression levels of miR-23a in all the groups were found. Myostatin, a negative regulator of skeletal muscle hypertrophy, was predicted as the target gene for miR-23a and miR-126-5p and we demonstrated their direct binding. Correlation analysis revealed association between miRNAs expression profiles and animals' weights along the age. Circulating miRNAs could be promising for future studies on their biomarker potentialities to beef cattle selection.

Liquid Biopsy for Cancer Cachexia: Focus on Muscle-Derived microRNAs.

Cancer cachexia displays a complex nature in which systemic inflammation, impaired energy metabolism, loss of muscle and adipose tissues result in unintentional body weight loss. Cachectic patients have a poor prognosis and the presence of cachexia reduces the tolerability of chemo/radio-therapy treatments and it is frequently the primary cause of death in advanced cancer patients. Early detection of this condition could make treatments more effective. However, early diagnostic biomarkers of cachexia are currently lacking. In recent years, although solid biopsy still remains the "gold standard" for diagnosis of cancer, liquid biopsy is gaining increasing interest as a source of easily accessible potential biomarkers. Moreover, the growing interest in circulating microRNAs (miRNAs), has made these molecules attractive for the diagnosis of several diseases, including cancer. Some muscle-derived circulating miRNA might play a pivotal role in the onset/progression of cancer cachexia. This topic is of great interest since circulating miRNAs might be easily detectable by means of liquid biopsies and might allow an early diagnosis of this syndrome. We here summarize the current knowledge on circulating muscular miRNAs involved in muscle atrophy, since they might represent easily accessible and promising biomarkers of cachexia.

Circulating Extracellular miRNA Analysis in Patients with Stable CAD and Acute Coronary Syndromes.

Extracellular circulating microRNAs (miRNAs) are currently a focus of interest as non-invasive biomarkers of cardiovascular pathologies, including coronary artery disease (CAD) and acute coronary syndromes (ACS): myocardial infarction with and without ST-segment elevation (STEMI and NSTEMI) and unstable angina (UA). However, the current data for some miRNAs are controversial and inconsistent, probably due to pre-analytical and methodological variances in different studies. In this work, we fulfilled the basic pre-analytical requirements provided for circulating miRNA studies for application to stable CAD and ACS research. We used quantitative PCR to determine the relative plasma levels of eight circulating miRNAs that are potentially associated with atherosclerosis. In a cohort of 136 adult clinic CAD patients and outpatient controls, we found that the plasma levels of miR-21-5p and miR-146a-5p were significantly elevated in ACS patients, and the level of miR-17-5p was decreased in ACS and stable CAD patients compared to both healthy controls and hypertensive patients without CAD. Within the ACS patient group, no differences were found in the plasma levels of these miRNAs between patients with positive and negative troponin, nor were any differences found between STEMI and NSTEMI. Our results indicate that increased plasma levels of miR-146a-5p and miR-21-5p can be considered general ACS circulating biomarkers and that lowered miR-17-5p can be considered a general biomarker of CAD.

Circulating miRNA is a useful diagnostic biomarker for nonalcoholic steatohepatitis in nonalcoholic fatty liver disease.

Nonalcoholic steatohepatitis (NASH) is considered as a progressive form of nonalcoholic fatty liver disease (NAFLD). To distinguish NASH from nonalcoholic fatty liver (NAFL), we evaluated the diagnostic value of circulating miRNAs. Small RNA sequencing was performed on 12 NAFL patients and 12 NASH patients, and the miRNA expression was compared. After selecting miRNAs for the diagnosis of NASH, we analyzed the diagnostic accuracy of each miRNA and the combination of miRNAs. External validation was performed using quantitative reverse transcription PCR. Among the 2,588 miRNAs, 26 miRNAs significantly increased in the NASH group than in the NAFL group. Among the 26 elevated miRNAs in the NASH group, 8 miRNAs were selected, and in silico analysis was performed. Only four miRNAs (miR-21-5p, miR-151a-3p, miR-192-5p, and miR-4449) showed significant area under the receiver operating characteristic curve (AUC) values for NASH diagnosis. The combination of the four miRNAs showed satisfactory diagnostic accuracy for NASH (AUC 0.875; 95% CI 0.676-0.973). External validation revealed similar diagnostic accuracy for NASH (AUC 0.874; 95% CI 0.724-0.960). NASH represents significantly distinct miRNA expression profile compared with NAFL. The combination of serum circulating miRNAs can be used as a novel biomarker for the NASH diagnosis in NAFLD.

Circulating miRNome of Trachemys scripta after elective gonadectomy under general anesthesia.

Post-surgical management is an important issue in veterinary medicine, requiring biomarkers with high sensitivity and specificity for timely and effective treatment. Emerging evidence suggests that miRNAs are promising stress- and pain-related markers. The aims were to profile the circulating miRNA signature in plasma of turtles (Trachemys scripta) and point out potential candidate biomarkers to assess the status of the animal. The plasma of female turtles underwent surgical gonadectomy were collected 24 h pre-surgery, and 2.5 h and 36 h post-surgery. The expression of miRNAs was profiled by Next Generation Sequencing and the dysregulated miRNAs were validated using RT-qPCR. The diagnostic value of miRNAs was calculated by ROC curves. The results showed that 14 miRNAs were differentially expressed over time. RT-qPCR validation highlighted that 2-miR-499-3p and miR-203-5p-out of 8 miRNAs tested were effectively modulated. The Area Under the Curve (AUC) of miR-203-5p was fair (AUC 0.7934) in discriminating pre- and 36 h post-surgery samples and poor for other time points; the AUC of miR-499-3p was excellent (AUC 0.944) in discriminating pre-surgery and 2.5 h post-surgery samples, and fair in discriminating pre-surgery and 36 h post-surgery (AUC 0.7292) and 2.5 h and 36 h post-surgery (AUC 0.7569) samples. In conclusion, we demonstrated for the first time that miRNAs profile changes in plasma of turtles underwent surgical oophorectomy and identified miR-203-5p and miR-499-3p as potential candidate biomarkers to assess animals' status. Further studies are necessary to confirm their diagnostic value and to investigate functional and mechanistic networks to improve our understanding of the biological processes.

Single-molecule amplification-free multiplexed detection of circulating microRNA cancer biomarkers from serum.

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.

Plasma Circulating Mirnas Profiling for Identification of Potential Breast Cancer Early Detection Biomarkers.

OBJECTIVE: This study aimed to characterize the miRNA expression profiles from plasma samples of our local breast cancer patients in comparison to healthy control by using miRNA PCR Array. METHODS: In this study, plasma miRNA profiles from eight early-stage breast cancer patients and nine age-matched (± 2 years) healthy controls were characterized by miRNA array-based approach, followed by differential gene expression analysis, Independent T-test and construction of Receiver Operating Characteristic (ROC) curve to determine the capability of the assays to discriminate between breast cancer and the healthy control. RESULTS: Based on the 372-miRNAs microarray profiling, a set of 40 differential miRNAs was extracted regarding to the fold change value at 2 and above. We further sub grouped 40 miRNAs of breast cancer patients that were significantly expressed at 2-fold change and higher. In this set, we discovered that 24 miRNAs were significantly upregulated and 16 miRNAs were significantly downregulated in breast cancer patients, as compared to the miRNA expression of healthy subjects. ROC curve analysis revealed that seven miRNAs (miR-125b-5p, miR-142-3p, miR-145-5p, miR-193a-5p, miR-27b-3p, miR-22-5p and miR-423-5p) had area under curve (AUC) value > 0.7 (AUC p-value < 0.05). Overlapping findings from differential gene expression analysis, ROC analysis, and Independent T-Test resulted in three miRNAs (miR-27b-3p, miR-22-5p, miR-145-5p). Cohen's effect size for these three miRNAs was large with d value are more than 0.95. CONCLUSION: miR-27b-3p, miR-22-5p, miR-145-5p could be potential biomarkers to distinguish breast cancer patients from healthy controls. A validation study for these three miRNAs in an external set of samples is ongoing.
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Urinary miRNA profiles discriminate between obstruction-induced bladder dysfunction and healthy controls.

Urgency, frequency and incomplete emptying are the troublesome symptoms often shared between benign prostatic obstruction-induced (BLUTD) and neurogenic (NLUTD) lower urinary tract dysfunction. Previously, using bladder biopsies, we suggested a panel of miRNA biomarkers for different functional phenotypes of the bladder. Urine is a good source of circulating miRNAs, but sex- and age-matched controls are important for urinary metabolite comparison. In two groups of healthy subjects (average age 32 and 57 years old, respectively) the total protein and RNA content was very similar between age groups, but the number of secreted extracellular vesicles (uEVs) and expression of several miRNAs were higher in the young healthy male volunteers. Timing of urine collection was not important for these parameters. We also evaluated the suitability of urinary miRNAs for non-invasive diagnosis of bladder outlet obstruction (BOO). A three urinary miRNA signature (miR-10a-5p, miR-301b-3p and miR-363-3p) could discriminate between controls and patients with LUTD (BLUTD and NLUTD). This panel of representative miRNAs can be further explored to develop a non-invasive diagnostic test for BOO. The age-related discrepancy in the urinary miRNA content observed in this study points to the importance of selecting appropriate, age-matched controls.

Postpartum circulating microRNA enhances prediction of future type 2 diabetes in women with previous gestational diabetes.

AIMS/HYPOTHESIS: Type 2 diabetes mellitus is a major cause of morbidity and death worldwide. Women with gestational diabetes mellitus (GDM) have greater than a sevenfold higher risk of developing type 2 diabetes in later life. Accurate methods for postpartum type 2 diabetes risk stratification are lacking. Circulating microRNAs (miRNAs) are well recognised as biomarkers/mediators of metabolic disease. We aimed to determine whether postpartum circulating miRNAs can predict the development of type 2 diabetes in women with previous GDM. METHODS: In an observational study, plasma samples were collected at 12 weeks postpartum from 103 women following GDM pregnancy. Utilising a discovery approach, we measured 754 miRNAs in plasma from type 2 diabetes non-progressors (n = 11) and type 2 diabetes progressors (n = 10) using TaqMan-based real-time PCR on an OpenArray platform. Machine learning algorithms involving penalised logistic regression followed by bootstrapping were implemented. RESULTS: Fifteen miRNAs were selected based on their importance in discriminating type 2 diabetes progressors from non-progressors in our discovery cohort. The levels of miRNA miR-369-3p remained significantly different (p < 0.05) between progressors and non-progressors in the validation sample set (n = 82; 71 non-progressors, 11 progressors) after adjusting for age and correcting for multiple comparisons. In a clinical model of prediction of type 2 diabetes that included six traditional risk factors (age, BMI, pregnancy fasting glucose, postpartum fasting glucose, cholesterol and triacylglycerols), the addition of the circulating miR-369-3p measured at 12 weeks postpartum improved the prediction of future type 2 diabetes from traditional AUC 0.83 (95% CI 0.68, 0.97) to an AUC 0.92 (95% CI 0.84, 1.00). CONCLUSIONS: This is the first demonstration of miRNA-based type 2 diabetes prediction in women with previous GDM. Improved prediction will facilitate early lifestyle/drug intervention for type 2 diabetes prevention.

MicroRNA Modulation of Host Immune Response and Inflammation Triggered by Helicobacter pylori.

Helicobacter pylori (H. pylori) remains the most-researched etiological factor for gastric inflammation and malignancies. Its evolution towards gastric complications is dependent upon host immune response. Toll-like receptors (TLRs) recognize surface and molecular patterns of the bacterium, especially the lipopolysaccharide (LPS), and act upon pathways, which will finally lead to activation of the nuclear factor-kappa B (NF-kB), a transcription factor that stimulates release of inflammatory cytokines. MicroRNAs (MiRNAs) finely modulate TLR signaling, but their expression is also modulated by activation of NF-kB-dependent pathways. This review aims to focus upon several of the most researched miRNAs on this subject, with known implications in host immune responses caused by H. pylori, including let-7 family, miRNA-155, miRNA-146, miRNA-125, miRNA-21, and miRNA-221. TLR-LPS interactions and their afferent pathways are regulated by these miRNAs, which can be considered as a bridge, which connects gastric inflammation to pre-neoplastic and malignant lesions. Therefore, they could serve as potential non-invasive biomarkers, capable of discriminating H. pylori infection, as well as its associated complications. Given that data on this matter is limited in children, as well as for as significant number of miRNAs, future research has yet to clarify the exact involvement of these entities in the progression of H. pylori-associated gastric conditions.

Subpopulations of exosomes purified via different exosomal markers carry different microRNA contents.

The heterogeneity of exosome populations presents a great challenge to their study. The current study was designed to investigate the potential heterogeneity miRNA contents in circulating exosomes purified via different exosomal markers. In this study, exosomes from the serum of C57BL/6 mice after cecum ligation and perforation (CLP) or sham operation were isolated by precipitation using ExoQuick-TC and affinity purified with anti-Rab5b, anti-CD9, anti-CD31, and anti-CD44 antibodies using the Exo-Flow Exosome Capture kit to collect exosome subpopulations. RNA extracted from the exosomes isolated by ExoQuick-TC were profiled by next-generation sequencing (NGS). Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was also employed to determine the expression profiles of four representative exosomal miRNAs (mmu-miR-486-5p, mmu-miR-10a-5p, mmu-miR-143-3p, and mmu-miR-25-3p) selected from the NGS analysis. The results revealed that the expression patterns of these miRNAs in exosomes isolated by ExoQuick-TC as determined by RT-qPCR and NGS were similar, showing upregulation of mmu-miR-10a-5p and mmu-miR-143-3p but downregulation of mmu-miR-25-3p and mmu-miR-486-5p following CLP when compared to the levels in exosomes from sham control mice. However, their expression levels in the antibody-captured exosome subpopulations varied. The miRNAs in the exosomes captured by anti-Rab5b or anti-CD9 antibodies were more similar to those isolated by ExoQuick-TC than to those captured by anti-CD44 antibodies. However, there were no significant differences in these four miRNAs in CD31-captured exosomes. This study demonstrated that purification with different exosomal markers allows the collection of different exosome subpopulations with various miRNA contents. The results of this study demonstrate the heterogeneity of circulating exosomes and suggest the importance of stratifying exosome subpopulations when using circulating exosomes as biomarkers or investigating exosome function. In addition, this study also emphasized the necessity of using a consistent exosome marker across different samples as detecting biomarkers.