Plasma Levels Of miRNAs Research Articles

Yigu decoction regulates plasma miRNA in postmenopausal osteoporosis patients: a randomized controlled trial.

Postmenopausal osteoporosis (PMOP) is a serious condition that affects elderly individuals. Our previous study revealed that Yigu decoction (YGD) effectively improved bone mineral density (BMD) in elderly individuals, but the mechanism underlying this effect remains unclear. In this study, we investigated the relationships among YGD, microRNAs (miRNAs), and bone metabolism by assessing the effects of YGD on the miRNA levels in patient plasma to provide a scientific basis for treating PMOP with YGD. In this clinical trial, 60 patients were randomly assigned to the YGD group or the control group (ratio of 1:1) and treated for 3months. The primary outcome measure was BMD, and the secondary outcome measures included plasma miRNA levels, visual analogue scale (VAS) scores, alkaline phosphatase (ALP) levels, anti-tartrate acid phosphatase (TRACP-5b) levels and traditional Chinese medicine (TCM) syndrome scores. We assessed the regulatory roles of miRNAs in PMOP patients by analysing publicly available data from the Gene Expression Omnibus (GEO) database. Bioinformatics methods were also used to explore the mechanism by which YGD regulates miRNAs that are involved in bone metabolism. Compared with those before treatment, the BMD, ALP levels, TRACP-5b levels, TCM syndrome scores and VAS scores improved in both groups after 3 months of treatment (P < 0.05). A total of 82 miRNAs differed between the groups. After analysing data from the GEO database, we confirmed that miR-133a-3p is the key molecule that mediates the effects of YGD intervention on PMOP. GO analysis of key genes suggested that gene enrichment was more pronounced in response to hormones, cellular response to growth factor stimulation, and positive regulation of physiological and metabolic processes. KEGG analysis revealed that these genes were enriched mainly in the PI3K-Akt, FOXO, and JAK-STAT pathways and other pathways. The results of the protein‒protein interaction (PPI) network analysis revealed that epidermal growth factor receptor (EGFR), Insulin-like growth factor 1 (IGF-1), Caveolin-1 (Cav-1) and others were core proteins. This study demonstrated that YGD is beneficial in the treatment of PMOP, ameliorating clinical symptoms and bone turnover indices. Moreover, the inhibition of miR-133a-3p expression may be the key mechanisms by which YGD regulates bone metabolism in the treatment of PMOP, although YGD regulates bone metabolism in a multitarget and multipathway manner.

Identification of Chronic Pancreatitis Associated microRNAs and Genes for the Diagnosis of Pancreatic Cancer.

The timely identification of both malignant and nonmalignant pancreatic lesions has the potential to significantly enhance prognosis and implement risk management strategies across various levels. microRNAs (miRs) and their corresponding targets play a crucial role in the development of pancreatic lesions and can serve as valuable diagnostic and therapeutic targets. The objective of our study was to investigate potential diagnostic markers that can effectively differentiate between malignant and nonmalignant pancreatic lesions. Gene Expression Omnibus (GEO) database with GSE24279 dataset was utilized to screen differentially expressed miRNAs (DEMs). We utilized the TargetScanHuman database to predict the target genes associated with hsa-miR-150-3p, hsa-miR-150-5p, and hsa-miR-214-3p. Furthermore, a cohort comprising healthy individuals (n = 52), chronic pancreatitis (CP; n = 34), and pancreatic adenocarcinoma (PAAD; n = 53) patients was recruited to ascertain the levels of plasma markers. We identified 3 miRNAs (hsa-miR-150-3p, hsa-miR-150-5p, and hsa-miR-214-3p) and 2 proteins (PCDH1 and AMN) as potential diagnostic markers for distinguishing between CP and PAAD. The area under the curve (AUC) values for all markers exceeded .800. Notably, a combination of plasma PCDH1 and AMN demonstrated excellent diagnostic performance (AUC = .921; 95% CI: .866-.977; sensitivity = .792; specificity = .941) in discriminating between CP and PAAD. In addition, the model of hsa-miR-150-3p, hsa-miR-150-5p, and hsa-miR-214-3p yielded an AUC of .928, sensitivity of .830, and specificity of .912, respectively. Plasma levels of miRNAs (hsa-miR-150-3p, hsa-miR-150-5p, and hsa-miR-214-3p) and their corresponding targets (PCDH1 and AMN) hold promise as potential biomarkers for predicting PAAD in patients with CP.

A comprehensive study of genetic regulation and disease associations of plasma circulatory microRNAs using population-level data.

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. Perturbations in plasma miRNA levels are known to impact disease risk and have potential as disease biomarkers. Exploring the genetic regulation of miRNAs may yield new insights into their important role in governing gene expression and disease mechanisms. We present genome-wide association studies of 2083 plasma circulating miRNAs in 2178 participants of the Rotterdam Study to identify miRNA-expression quantitative trait loci (miR-eQTLs). We identify 3292 associations between 1289 SNPs and 63 miRNAs, of which 65% are replicated in two independent cohorts. We demonstrate that plasma miR-eQTLs co-localise with gene expression, protein, and metabolite-QTLs, which help in identifying miRNA-regulated pathways. We investigate consequences of alteration in circulating miRNA levels on a wide range of clinical conditions in phenome-wide association studies and Mendelian randomisation using the UK Biobank data (N = 423,419), revealing the pleiotropic and causal effects of several miRNAs on various clinical conditions. In the Mendelian randomisation analysis, we find a protective causal effect of miR-1908-5p on the risk of benign colon neoplasm and show that this effect is independent of its host gene (FADS1). This study enriches our understanding of the genetic architecture of plasma miRNAs and explores the signatures of miRNAs across a wide range of clinical conditions. The integration of population-based genomics, other omics layers, and clinical data presents opportunities to unravel potential clinical significance of miRNAs and provides tools for novel miRNA-based therapeutic target discovery.

MicroRNAs are enriched at COVID-19 genomic risk regions, and their blood levels correlate with the COVID-19 prognosis of cancer patients infected by SARS-CoV-2

BackgroundCancer patients are more susceptible to an aggressive course of COVID-19. Developing biomarkers identifying cancer patients at high risk of COVID-19-related death could help determine who needs early clinical intervention. The miRNAs hosted in the genomic regions associated with the risk of aggressive COVID-19 could represent potential biomarkers for clinical outcomes.Patients and methodsPlasma samples were collected at The University of Texas MD Anderson Cancer Center from cancer patients (N = 128) affected by COVID-19. Serum samples were collected from vaccinated healthy individuals (n = 23) at the Municipal Clinical Emergency Teaching Hospital in Timisoara, Romania. An in silico positional cloning approach was used to identify the presence of miRNAs at COVID-19 risk-associated genomic regions: CORSAIRs (COvid-19 RiSk AssocIated genomic Regions). The miRNA levels were measured by RT-qPCR.ResultsWe found that miRNAs were enriched in CORSAIR. Low plasma levels of hsa-miR-150-5p and hsa-miR-93-5p were associated with higher COVID-19-related death. The levels of hsa-miR-92b-3p were associated with SARS-CoV-2 test positivity. Peripheral blood mononuclear cells (PBMC) increased secretion of hsa-miR-150-5p, hsa-miR-93-5p, and hsa-miR-92b-3p after in vitro TLR7/8- and T cell receptor (TCR)-mediated activation. Increased levels of these three miRNAs were measured in the serum samples of healthy individuals between one and nine months after the second dose of the Pfizer-BioNTech COVID-19 vaccine. SARS-CoV-2 infection of human airway epithelial cells influenced the miRNA levels inside their secreted extracellular vesicles.ConclusionsMiRNAs are enriched at CORSAIR. Plasma miRNA levels can represent a potential blood biomarker for predicting COVID-19-related death in cancer patients.

Circulating microRNAs and isomiRs as biomarkers for the initial insult and epileptogenesis in four experimental epilepsy models: The EPITARGET study.

Structural epilepsies can manifest months or years after the occurrence of an initial epileptogenic insult, making them amenable for secondary prevention. However, development of preventive treatments has been challenged by a lack of biomarkers for identifying the subset of individuals with the highest risk of epilepsy after the epileptogenic insult. Four different rat models of epileptogenesis were investigated to identify differentially expressed circulating microRNA (miRNA) and isomiR profiles as biomarkers for epileptogenesis. Plasma samples were collected on day 2 and day 9 during the latency period from animals that did or did not develop epilepsy during long-term video-electroencephalographic monitoring. miRNAs and isomiRs were identified and measured in an unsupervised manner, using a genome-wide small RNA sequencing platform. Receiver operating characteristic analysis was performed to determine the performance of putative biomarkers. Two days after an epileptogenic insult, alterations in the levels of several plasma miRNAs and isomiRs predicted epileptogenesis in a model-specific manner. One miRNA, miR-3085, showed good sensitivity (but low specificity) as a prognostic biomarker for epileptogenesis in all four models (area under the curve = .729, sensitivity = 83%, specificity = 64%, p < .05). Identified plasma miRNAs and isomiRs are mostly etiology-specific rather than common prognostic biomarkers of epileptogenesis. These data imply that in preclinical and clinical studies, it may be necessary to identify specific biomarkers for different epilepsy etiologies. Importantly, circulating miRNAs like miR-3085 with high negative predictive value for epileptogenesis in different etiologies could be useful candidates for initial screening purposes of epileptogenesis risk.

Over-Expression of MicroRNA-210 and MicroRNA-185-5p in Normotensive Late-Fetal Growth Restriction: Preliminary Cohort Study

OBJECTIVE: Late Fetal Growth Restriction (LFGR), a condition associated with perinatal and long-term neurodevelopmental problems, is caused by inadequate placental transfer of oxygen and nutrients to the fetus. This preliminary study evaluates the expression profiles and clinical significance of hypoxia-sensitive microRNA-210 (miR-210) and microRNA-185-5p (miR-185-5p) in placental and maternal circulation. The potential clinical implications of this research could significantly enhance the management of LFGR. STUDY DESIGN: In this prospective cohort study conducted at the Gynecology and Obstetrics Clinic of Trakya University Faculty of Medicine Hospital, miR-210 and miR-185-5p expression was evaluated in the placenta of healthy (n=30) and LFGR (n=30) cases. In healthy (n=15) and LFGR (n=15) cases, miRNA levels in maternal plasma were studied. We determined these two miRNAs using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) and statistically analyzed them with clinical data to determine robustness. We provided a comprehensive evaluation. RESULTS: Both the placenta and maternal plasma samples exhibited significant correlations with miR-210 and miR-185-5p expression (r=0.615; r=0.771, p&lt;0.01). The expression levels in both placenta and plasma were significantly higher in the LFGR group. Placental miR-210 demonstrated an AUC (0.908), sensitivity (93.3%), and specificity (96.7%), while plasma miR-210 showed AUC (0.738), sensitivity (86.7%), and specificity (66.7%). In contrast, placental miRNA 185-5p displayed a diagnostic performance with AUC (0.926), sensitivity (76.1%), and specificity (63.8%); plasma miRNA 185-5p exhibited an AUC (0.778), sensitivity (86.7%), and specificity (86.7%). However, placental miR-210 and miR-185-5p did not fully correlate with birth weight, placental weight, and fetal cerebroplacental Doppler ratio. CONCLUSION: Placental miRNA-210 showed superior diagnostic performance over placental miRNA 185-5p in LFGR pregnancies. However, it is crucial to emphasize that further studies are needed to validate these findings and correlate them with clinical data, underscoring the ongoing importance of research in this field.

Early microRNA and metabolite changes after anterior cruciate ligament reconstruction surgery

ObjectivesAnterior cruciate ligament (ACL) reconstruction after injury does not prevent post-traumatic osteoarthritis (PTOA). Circulating microRNA (miRNA) and metabolite changes emerging shortly after ACL injury and reconstruction remain insufficiently defined, potentially harbouring early cues contributing to PTOA evolution. Moreover, their differential expression between females and males also may influence PTOA’s natural trajectory. This study aims to determine alterations in plasma miRNA and metabolite levels in the early stages following ACL reconstruction and between females and males. MethodsA cohort of 43 ACL reconstruction patients was examined. Plasma was obtained at baseline, 2-weeks, and 6-weeks post-surgery (129 biospecimens in total). High throughput miRNA sequencing and metabolomics were conducted. Differentially expressed miRNAs and metabolites were identified using negative binomial and linear regression models, respectively. Associations between miRNAs and metabolites were explored using time and sex as co-variants, (pre- versus 2- and 6-weeks post-surgery). Using computational biology, miRNA-metabolite-gene interaction and pathway analyses were performed. ResultsLevels of 46 miRNAs were increased at 2-weeks post-surgery compared to pre-surgery (baseline) using miRNA sequencing. Levels of 13 metabolites were significantly increased while levels of 6 metabolites were significantly decreased at 2-weeks compared to baseline using metabolomics. Hsa-miR-145-5p levels were increased in female subjects at both 2-weeks (log2-fold-change 0.71, 95%CI 0.22,1.20) and 6-weeks (log2-fold-change 0.75, 95%CI 0.07,1.43) post-surgery compared to males. In addition, hsa-miR-497-5p showed increased levels in females at 2-weeks (log2-fold-change 0.77, 95%CI 0.06,1.48) and hsa-miR-143-5p at 6-weeks (log2-fold-change 0.83, 95%CI 0.07,1.59). Five metabolites were decreased at 2-weeks post-surgery in females compared to males: L-leucine (-1.44, 95%CI -1.75,-1.13), g-guanidinobutyrate (-1.27, 95%CI 1.54,-0.99), creatinine (-1.17, 95%CI -1.44,-0.90), 2-methylbutyrylcarnitine (-1.76, 95%CI -2.17,-1.35), and leu-pro (-1.13, 95%CI -1.44,-0.83). MiRNA-metabolite-gene interaction analysis revealed key signalling pathways based on post-surgical time-point and in females versus males. ConclusionMiRNA and metabolite profiles were modified by time and by sex early after ACL reconstruction surgery, which could influence surgical response and ultimately risk of developing PTOA.

Exposure to per- and polyfluoroalkyl substances and alterations in plasma microRNA profiles in children

BackgroundPer- and polyfluoroalkyl substances (PFAS) are synthetic chemicals that persist in the environment and can accumulate in humans, leading to adverse health effects. MicroRNAs (miRNAs) are emerging biomarkers that can advance the understanding of the mechanisms of PFAS effects on human health. However, little is known about the associations between PFAS exposures and miRNA alterations in humans. ObjectiveTo investigate associations between PFAS concentrations and miRNA levels in children. MethodsData from two distinct cohorts were utilized: 176 participants (average age 17.1 years; 75.6% female) from the Teen-Longitudinal Assessment of Bariatric Surgery (Teen-LABS) cohort in the United States, and 64 participants (average age 6.5 years, 39.1% female) from the Rhea study, a mother-child cohort in Greece. PFAS concentrations and miRNA levels were assessed in plasma samples from both studies. Associations between individual PFAS and plasma miRNA levels were examined after adjusting for covariates. Additionally, the cumulative effects of PFAS mixtures were evaluated using an exposure burden score. Ingenuity Pathways Analysis was employed to identify potential disease functions of PFAS-associated miRNAs. ResultsPlasma PFAS concentrations were associated with alterations in 475 miRNAs in the Teen-LABs study and 5 miRNAs in the Rhea study (FDR p < 0.1). Specifically, plasma PFAS concentrations were consistently associated with decreased levels of miR-148b-3p and miR-29a-3p in both cohorts. Pathway analysis indicated that PFAS-related miRNAs were linked to numerous chronic disease pathways, including cardiovascular diseases, inflammatory conditions, and carcinogenesis. ConclusionThrough miRNA screenings in two independent cohorts, this study identified both known and novel miRNAs associated with PFAS exposure in children. Pathway analysis revealed the involvement of these miRNAs in several cancer and inflammation-related pathways. Further studies are warranted to enhance our understanding of the relationships between PFAS exposure and disease risks, with miRNA emerging as potential biomarkers and/or mediators in these complex pathways.

Plasma Extracellular MicroRNAs Associated With Cardiovascular Disease Risk Factors in Middle-Aged and Older Adults.

Extracellular microRNAs (miRNAs) are a class of noncoding RNAs that remain stable in the extracellular milieu, where they contribute to various physiological and pathological processes by facilitating intercellular signaling. Previous studies have reported associations between miRNAs and cardiovascular diseases (CVDs); however, the plasma miRNA signatures of CVD and its risk factors have not been fully elucidated at the population level. Plasma miRNA levels were measured in 4440 FHS (Framingham Heart Study) participants. Linear regression analyses were conducted to test the cross-sectional associations of each miRNA with 8 CVD risk factors. Prospective analyses of the associations of miRNAs with new-onset obesity, hypertension, type 2 diabetes, CVD, and all-cause mortality were conducted using proportional hazards regression. Replication was carried out in 1999 RS (Rotterdam Study) participants. Pathway enrichment analyses were conducted and target genes were predicted for miRNAs associated with ≥5 risk factors in the FHS. In the FHS, 6 miRNAs (miR-193b-3p, miR-122-5p, miR-365a-3p, miR-194-5p, miR-192-5p, and miR-193a-5p) were associated with ≥5 risk factors. This miRNA signature was enriched for pathways associated with CVD and several genes annotated to these pathways were predicted targets of the identified miRNAs. Furthermore, miR-193b-3p, miR-194-5p, and miR-193a-5p were each associated with ≥2 risk factors in the RS. Prospective analysis revealed 8 miRNAs associated with all-cause mortality in the FHS. These findings highlight associations between miRNAs and CVD risk factors that may provide valuable insights into the underlying pathogenesis of CVD.

Preanalytical considerations for clinical assays of circulating human miRNA-451a, miRNA-423-5p and miRNA-199a-3p for diagnostic purposes.

Circulating miRNA has recently emerged as important biomolecules with potential clinical values as diagnostic markers for several diseases. However, to be used as such, it is critical to accurately quantify miRNAs in the clinic. Yet, preanalytical factors that can affect an error-free quantification of these miRNAs have not been explored. This study aimed at investigating several of these preanalytical factors that may affect the accurate quantification of miRNA-451a, miRNA-423-5p and miRNA-199a-3p in human blood samples. We initially evaluated levels of these three miRNAs in red blood cells (RBCs), white blood cells (WBCs), platelets, and plasma by droplet digital PCR (ddPCR). Next, we monitored miRNA levels in whole blood or platelet rich plasma (PRP) stored at different temperatures for different time periods by ddPCR. We also investigated the effects of hemolysis on miRNA concentrations in platelet-free plasma (PFP). Our results demonstrate that more than 97% of miRNA-451a and miRNA-423-5p in the blood are localized in RBCs, with only trace amounts present in WBCs, platelets, and plasma. Highest amount of the miRNA-199a-3p is present in platelets. Hemolysis had a significant impact on both miRNA-451a and miRNA-423-5p concentrations in plasma, however miRNA-199a levels remain unaffected. Importantly, PRP stored at room temperature (RT) or 4°C showed a statistically significant decrease in miRNA-451a levels, while the other two miRNAs were increased, at days 1, 2, 3 and 7. PFP at RT caused statistically significant steady decline in miRNA-451a and miRNA-423-5p, observed at 12, 24, 36, 48 and 72 hours. Levels of the miRNA-199a-3p in PFP was stable during first 72 hours at RT. PFP stored at -20°C for 7 days showed declining stability of miRNA-451a over time. However, at -80°C miRNA-451a levels were stable up to 7 days. Together, our data indicate that hemolysis and blood storage at RT, 4°C and -20°C may have significant negative effects on the accuracy of circulating miRNA-451a and miRNA-423-5p quantification.

No Detectable Differences in microRNA Plasma Levels between Diabetic Hypertensive Patients with and without Incident Subclinical Atrial Fibrillation.

Background: Long-term rhythm monitoring (LTRM) can detect undiagnosed atrial fibrillation (AF) in patients at risk of AF and stroke. Circulating microRNAs (miRNAs), which have been shown to play a role in atrial electrical and structural remodelling, could help to select patients who would benefit most from LTRM. The aim of this study was to investigate whether patients with diabetes mellitus (DM) and hypertension and screen-detected subclinical AF (SCAF) using an insertable cardiac monitor (ICM) have significantly different plasma baseline levels of five selected miRNAs playing a role in the modulation of atrial electrical and structural remodelling (miR-21-5p, miR-29b-3p, miR-150-5p, miR-328-3p, and miR-432-5p) compared to those without SCAF. Methods: This study was performed at the outpatient clinic of a secondary academic teaching hospital between December 2013 and November 2015. Eligible patients were ≥65 years of age with DM and hypertension but without known heart diseases. All patients received an ICM. On the day of ICM implantation, blood samples for the measurement of plasma levels of the five miRNAs were drawn. In this post hoc analysis, we investigated their expression by reverse transcription-quantitative polymerase chain reaction. MiRNA plasma levels in patients with and without newly detected SCAF were compared. Results: We included 82 consecutive patients (median age of 71.3 years (IQR 67.4-75.1)), who were followed for a median of 588 days (IQR: 453-712 days). Seventeen patients (20.7%) had ICM-detected SCAF. Plasma levels of miR-328-3p, miR-29b-3p, miR-21-5p, miR-432-5p, and miR-150-5p were slightly but not significantly different in patients with incident SCAF compared with patients without. Conclusions: In patients with hypertension and DM, newly detected SCAF was not significantly associated with changes in expression levels of miR-21-5p, miR-29b-3p, miR-150-5p, miR-328-3p, and miR-432-5p.

Differences in serum and plasma levels of microRNAs and their time-course changes after blood collection

BackgroundSerum and plasma are used for measurements of microRNAs (miRNAs) as biomarkers of various diseases. However, no consistent findings have been obtained regarding differences in serum and plasma levels of miRNAs. The purpose of this study was to clarify differences in serum and plasma levels of total miRNAs and their time-course changes after blood collection. MethodsVenous blood was collected from healthy men, and samples were prepared at the time points of 0, 15, 30, 60 and 180 min after blood collection for plasma and after clot formation for serum. Levels of total miRNAs were analyzed by the hybridization method using the 3D-Gene miRNA Oligo chip. ResultsAbout one third of 2632 miRNAs tested showed levels high enough for comparison of serum and plasma levels and for investigation of their time-course changes. Levels of 299 miRNAs at time 0 were significantly different in serum and plasma. Levels of representative platelet-derived miRNAs including miR-185-5p, -22-3p and -320b were significantly higher in plasma than in serum, while levels of representative erythrocyte-derived miRNAs including miR-451a, -486-5p and -92a-3p were not significantly different in serum and plasma. Plasma levels of 173 miRNAs and 6 miRNAs showed significant decreasing and increasing tendencies, respectively, while there were no miRNAs in serum that showed significant time-course changes. ConclusionThe results suggest that careful attention should be paid when comparing serum and plasma levels of miRNAs and that plasma samples should be prepared early after blood collection.

ASSESSMENT OF CIRCULATING miRNA LEVELS IN BREAST CANCER PATIENTS DEPENDING ON CLINICAL CHARACTERISTICS AND CHEMOTHERAPY.

Breast cancer (BC) stands out as the most prevalent cancer in women. The levels of miRNA expression before and after chemotherapy are considered a potential indicator for the prognosis of the disease. To study blood plasma miRNA levels in BC patients and to assess their correlation with the menopausal status, disease stage, and molecular BC subtype. Blood plasma levels of 6 miRNAs (miRNA-25, miRNA-27, miRNA-155, miRNA-200, miRNA-335, and miRNA-497) were studied in 70 BC patients and 18 healthy individuals using RT-PCR. miRNA-25, miRNA-335, and miRNA-497 levels were significantly higher in BC patients, while a tendency toward a decrease in the miRNA-27 and miRNA-335 levels in premenopausal patients and high miRNA-27 levels in menopausal patients was established. After neoadjuvant chemotherapy, a decrease in the miRNA-25 and miRNA-335 levels was registered. The results indicated that miRNA-25, miRNA-27, miRNA-335, and miRNA-497 deserve attention as markers for assessing the efficacy of treatment of BC patients.

Circulating MicroRNAs as potential biomarkers for cerebral collateral circulation in symptomatic carotid stenosis.

Cerebral collateral circulation (CCC) considerably improves the prognosis of patients with symptomatic carotid stenosis (SCS). This study evaluated the diagnostic value of plasma microRNAs (miRNAs) in determining CCC status in patients with SCS. This single-center observational study enrolled patients with ≥50% carotid artery stenosis diagnosed using Doppler ultrasound. CCC was assessed using cerebrovascular digital subtraction angiography (DSA). Quantitative reverse transcription-polymerase chain reaction was used to determine the expression levels of plasma miRNAs. A multivariate logistic regression model and receiver operating characteristic (ROC) curve were used to analyze the diagnostic value of plasma miRNA expression in determining CCC status. A total of 43 patients were enrolled (28 with CCC and 15 without CCC). The plasma expression levels of miR-126-3p, miR-132-3p, and miR-210-3p were significantly higher and those of miR-16-3p and miR-92-3p were significantly lower in patients with CCC. After adjusting for age, gender, drinking history, comorbidities and degree of SCS, miR-92a-3p, miR-126-3p, miR-132-3p, and miR-210-3p were found to be significantly associated with CCC establishment (p < 0.05). ROC curve analysis indicated a high diagnostic value of these miRNAs in determining CCC status [area under the curve (AUC): 0.918-0.965], with miR-126-3p exhibiting the highest predictive performance (AUC: 0.965). Subgroup analysis revealed that patients with CCC who had 50%-70% stenosis showed significantly higher expression level of miR-126-3p, whereas those with CCC who had 70%-99% stenosis showed significantly higher expression levels of miR-126-3p, miR-132-3p, and miR-210-3p as well as significantly lower expression levels of miR-15a-3p, miR-16-3p, and miR-92a-3p. The results indicate that these six plasma miRNAs have promising diagnostic value in determining CCC status in varying degrees of SCS. These miRNAs can serve as biomarkers for CCC status following SCS, with miR-126-3p showing the strongest positive correlation.

Growth-suppressor microRNAs mediate synaptic overgrowth and behavioral deficits in Fragile X mental retardation protein deficiency

Abnormal neuronal and synapse growth is a core pathology resulting from deficiency of the Fragile X mental retardation protein (FMRP), but molecular links underlying the excessive synthesis of key synaptic proteins remain incompletely defined. We find that basal brain levels of the growth suppressor let-7 microRNA (miRNA) family are selectively lowered in FMRP-deficient mice and activity-dependent let-7 downregulation is abrogated. Primary let-7 miRNA transcripts are not altered in FMRP-deficiency and posttranscriptional misregulation occurs downstream of MAPK pathway induction and elevation of Lin28a, a let-7 biogenesis inhibitor. Neonatal restoration of brain let-7 miRNAs corrects hallmarks of FMRP-deficiency, including dendritic spine overgrowth and social and cognitive behavioral deficits, in adult mice. Blockade of MAPK hyperactivation normalizes let-7 miRNA levels in both brain and peripheral blood plasma from Fmr1 KO mice. These results implicate dysregulated let-7 miRNA biogenesis in the pathogenesis of FMRP-deficiency, and highlight let-7 miRNA-based strategies for future biomarker and therapeutic development.

Plasma microrna quantification protocol.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate translation and are involved in many pathological processes. They have emerged as promising biomarkers for diagnosis of conditions such as aortic aneurysm disease. Quantifying miRNAs in plasma is uniquely challenging because of the lack of standardized reproducible protocols. To facilitate the independent verification of conclusions, it is necessary to provide a thorough disclosure of all pertinent experimental details. In this technical note, we present a comprehensive protocol for quantifying plasma miRNAs using droplet digital PCR. We detail the entire workflow, including blood collection, plasma processing, cryo-storage, miRNA isolation, reverse transcription, droplet generation, PCR amplification, fluorescence reading, and data analysis. We offer comprehensive guidance regarding optimization, assay conditions, expected results, and insight into the troubleshooting of common issues. The stepwise normalization and detailed methodological guide enhance reproducibility. Moreover, multiple portions of this protocol may be automated. The data provided in this technical note is demonstrative of the values typically obtained when following its steps. To facilitate standardization in data reporting, we include a table of expected aortic aneurysm-related miRNA levels in healthy human plasma. This versatile protocol can be easily adapted to quantify most circulating miRNAs in plasma, making it a valuable resource for diagnostic development.

Abstract 13280: Plasma Extracellular microRNAs Associated With Cardiovascular Disease Risk Factors in Middle-Aged and Older Populations

Background: Extracellular microRNAs (miRNAs) are a class of non-coding RNAs that resist degradation and remain stable in the extracellular milieu where they contribute to various physiological and pathological processes by facilitating intercellular signaling. Previous studies have reported associations between miRNAs and cardiovascular diseases (CVD); however, the plasma miRNA signatures of CVD and its risk factors have not been fully elucidated at the population level. Methods: Plasma miRNA levels were measured in 4,440 Framingham Heart Study (FHS) participants from the Third Generation and Omni II cohorts using qRT-PCR. Linear regression analyses were conducted to test the cross-sectional associations of each miRNA with CVD risk factors, including systolic blood pressure (SBP), diastolic blood pressure (DBP), triglycerides, HDL and total cholesterol, blood glucose, BMI, and cigarette smoking. Prospective analyses of the associations of miRNAs with new-onset obesity, hypertension, type 2 diabetes, CVD, and all-cause mortality were conducted using proportional hazards regression. A false discovery rate threshold of 0.05 was applied for each risk factor and outcome individually. Replication was carried out in 2,000 Rotterdam Study (RS) participants with data on plasma miRNA levels measured using RNA-seq. Pathway enrichment analyses were conducted and gene targets were predicted for miRNAs associated with five or more risk factors in FHS. Results and Conclusions: In FHS, six miRNAs (miR-193b-3p, miR-122-5p, miR-365a-3p, miR-194-5p, miR-192-5p, miR-193a-5p) were associated with five or more CVD risk factors. This miRNA signature was enriched for biological processes and cellular pathways associated with CVD; many of the genes annotated to these pathways are predicted targets of the identified miRNAs. In RS, miR-193b-3p, miR-194-5p, and miR-193a-5p were associated with two or more risk factors. Prospective analysis revealed eight miRNAs associated with all-cause mortality only in FHS, including miR-193b-3p, miR-122-5p, miR-192-5p, and miR-193a-5p. These findings suggest plasma miRNA levels are associated with CVD risk factors and could provide opportunities to investigate their possible contributions to CVD pathogenesis.

Brain cell-specific origin of circulating microRNA biomarkers in experimental temporal lobe epilepsy.

The diagnosis of epilepsy is complex and challenging and would benefit from the availability of molecular biomarkers, ideally measurable in a biofluid such as blood. Experimental and human epilepsy are associated with altered brain and blood levels of various microRNAs (miRNAs). Evidence is lacking, however, as to whether any of the circulating pool of miRNAs originates from the brain. To explore the link between circulating miRNAs and the pathophysiology of epilepsy, we first sequenced argonaute 2 (Ago2)-bound miRNAs in plasma samples collected from mice subject to status epilepticus induced by intraamygdala microinjection of kainic acid. This identified time-dependent changes in plasma levels of miRNAs with known neuronal and microglial-cell origins. To explore whether the circulating miRNAs had originated from the brain, we generated mice expressing FLAG-Ago2 in neurons or microglia using tamoxifen-inducible Thy1 or Cx3cr1 promoters, respectively. FLAG immunoprecipitates from the plasma of these mice after seizures contained miRNAs, including let-7i-5p and miR-19b-3p. Taken together, these studies confirm that a portion of the circulating pool of miRNAs in experimental epilepsy originates from the brain, increasing support for miRNAs as mechanistic biomarkers of epilepsy.

Circulating miR-3613-5p but not miR-125b-5p, miR-199a-3p, and miR-451a are biomarkers of endometriosis

ObjectiveThis study aimed to assess the utility of circulating miR-125b-5p, miR-199a-3p, miR-451a, and miR-3613-5p as biomarkers of endometriosis. Study designPatients with stage III or IV of endometriosis according to the revised American Society of Reproductive Medicine (rASRM) staging classification, as well as control women, were recruited. We created a prospective study conducted on a group of 48 patients (n = 25 controls, n = 24 endometriosis) who had laparoscopic surgery. Blood samples were taken and plasma miRNA levels were measured by quantitative real-time polymerase chain reaction (RT-qPCR) and assessed with AUC and ROC curves. ResultsMiR-451a and miR-3613-5p were significantly decreased in the plasma of endometriosis patients. miR-451a had a receiver-operating characteristic (ROC) area under the curve 0.8283 and miR-3613-5p had a ROC area under the curve 0.7617. The concentration of circulating miR-125b-5p and miR-199-3p did not differ between endometriosis patients and controls. Plasma miRNA levels did not change with BMI, smoking status, fertility problems, or menstrual pain according to the VAS scale (p > 0.05). ConclusionCirculating miR-451a and miR-3613-5p levels significantly differed between endometriosis and controls. However, the levels of miR-451a were discordant with previous studies. Therefore, miR-3613-5p may have better potential as the endometriosis biomarker. Circulating miR-125b-5p and miR-199a-3p cannot be used as reliable markers of endometriosis.

Association between blood miR-26a levels following artificial insemination, and pregnancy outcome in dairy cattle.

Early pregnancy diagnosis is key to maximise productivity of dairy herds. We previously showed that an increase in the levels of miR-26 could be detected as soon as day 8 of pregnancy in heifers. The aims of this study were to determine whether 1) plasma miR-26 levels would be distinctly elevated, retrospectively, early after artificial insemination in lactating cows with successful compared to failed pregnancies, 2) the early increase in miRNA levels in cows with successful pregnancy could be accounted for by changes in miRNA expression in white blood cells (WBCs), presumably induced by the effects of embryo-derived interferon tau (IFNt), and 3) plasma miRNA levels may provide a reliable early predictor of pregnancy that could be used at a herd level. Blood samples were taken from a total of 34 dairy cows (lactation number 1 to 4) before (D0) and 9 and 18 days after artificial insemination at oestrus, followed by confirmation of pregnancy status by ultrasound on D32. In addition, WBCs collected from non-pregnant cows (n = 4) were stimulated in vitro with recombinant ovine IFNt (0-100 pg/ml). Levels of miRNAs and ISG15, a known IFNt-induced gene, were quantified by qPCR. Relative to D0, a larger increase in plasma miR-26a (P = 0.04) occurred on D9 in cows later confirmed to be pregnant (n = 12) than in cows with a failed pregnancy (n = 22). Expression of miR-26a in WBCs was not affected (P>0.1) by pregnancy status or IFNt stimulation in vitro, in contrast to ISG15 expression which increased markedly (P<0.0001) both in WBC samples collected on D18 from animals later confirmed to be pregnant, and in WBCs after stimulation with IFNt in vitro. Finally, ROC analyses revealed that miR-26a on D9 or D18 could predict pregnancy outcome with much lower accuracy than WBC ISG15 on D18 (Likelihood ratio, 2.3 vs 15.4). In summary, a modest increase in plasma miR-26a levels occurs during early pregnancy in mature dairy cows which may not accounted for by changes in miRNA levels in WBCs or the effects of IFNt. Moreover, compared to ISG15, changes in miR-26a levels may not provide an accurate test for early diagnosis of pregnancy in cows.