Plasma levels of microRNA-24, microRNA-320a, and microRNA-423-5p are potential biomarkers for colorectal carcinoma.
ABSTRACT: MicroRNAs are stable and easy to detect in plasma. The plasma levels of microRNAs are often changed in disease conditions, including cancer. This makes circulating microRNAs a novel class of biomarkers for cancer diagnosis. Analyses of online microRNA data base revealed that expression level of three microRNAs, microRNA-24 (miR-24), microRNA-320a (miR-320a), and microRNA-423-5p (miR-423-5p) were down-regulated in colorectal cancer (CRC). However, whether the plasma level of these three microRNAs can serve as biomarkers for CRC diagnosis and prognosis is not determined.Plasma samples from 223 patients with colorectal related diseases (111 cancer carcinoma, 59 adenoma, 24 colorectal polyps and 29 inflammatory bowel disease) and 130 healthy controls were collected and subjected to reverse transcription-quantitative real time PCR (RT-qPCR) analyses for the three microRNAs. In addition, plasma samples from 43 patients were collected before and after surgical treatment for the same RT-qPCR analyses.The concentrations of plasma miR-24, miR-320a and miR-423-5p were all decreased in patients with CRC and benign lesions (polyps and adenoma) compared with healthy controls, but increased in inflammatory bowel disease (IBD). The sensitivity of miR-24, miR-320a and miR-423-5p for early stage of CRC were 77.78 %, 90.74 %, and 88.89 %, respectively. Moreover, the plasma concentration of the three microRNAs was increased in patients after the surgery who had clinical improvement.The plasma levels of miR-24, miR-320a, and miR-423-5p have promising potential to serve as novel biomarkers for CRC detection, especially for early stage of CRC, which are superior to the currently used clinical biomarkers for CRC detection, such as CEA and CA19-9. Further efforts to develop the three microRNAs as biomarkers for early CRC diagnosis and prediction of surgical treatment outcomes are warrant.
Project description:MicroRNAs play functional roles in the etiology of type 2 diabetes mellitus (T2DM) and complications, and extracellular microRNAs have attracted interest as potential biomarkers of these conditions. We aimed to identify a set of plasma microRNAs, which could serve as biomarkers of T2DM and complications in a mixed Israeli Arab/Jewish patient sample. Subjects included 30 healthy volunteers, 29 early-stage T2DM patients, and 29 late-stage T2DM patients with renal and/or vascular complications. RNA was isolated from plasma, and the levels of 12 candidate microRNAs were measured by quantitative reverse transcription and polymerase chain reaction (qRT-PCR). MicroRNA levels were compared between the groups and correlated to clinical measurements, followed by stepwise regression analysis and discriminant analysis. Plasma miR-486-3p and miR-423 were respectively up- and down-regulated in T2DM patients compared to healthy controls. MiR-28-3p and miR-423 were up-regulated in patients with complicated T2DM compared to early T2DM, while miR-486-3p was down-regulated. Combined, four microRNAs (miR-146a-5p, miR-16-2-3p, miR-126-5p, and miR-30d) could distinguish early from complicated T2DM with 77% accuracy and 79% sensitivity. In male patients only, the same microRNAs, with the addition of miR-423, could distinguish early from complicated T2DM with 83.3% accuracy. Furthermore, plasma microRNA levels showed significant correlations with clinical measurements, and these differed between men and women. Additionally, miR-183-5p levels differed significantly between the ethnic groups. Our study identified a panel of specific plasma microRNAs which can serve as biomarkers of T2DM and its complications and emphasizes the importance of sex differences in their clinical application.
Project description:Blood-derived microRNA signatures have emerged as powerful biomarkers for predicting and diagnosing cardiovascular disease, cancer, and metabolic disorders. Platelets and platelet-derived microvesicles are a major source of microRNAs. We have previously shown that the inappropriate anticoagulation and storage of blood samples causes substantial platelet activation that is associated with the release of platelet-stored molecules into the plasma. However, it is currently unclear if circulating microRNA levels are affected by artificial platelet activation due to suboptimal plasma preparation. To address this issue, we used a standardized RT-qPCR test for 12 microRNAs (thrombomiR®, TAmiRNA GmbH, Vienna, Austria) that have been associated with cardiovascular and thrombotic diseases and were detected in platelets and/other hematopoietic cells. Blood was prevented from coagulating with citrate-theophylline-adenosine-dipyridamole (CTAD), sodium citrate, or ethylenediaminetetraacetic acid (EDTA) and stored for different time periods either at room temperature or at 4 °C prior to plasma preparation and the subsequent quantification of microRNAs. We found that five microRNAs (miR-191-5p, miR-320a, miR-21-5p, miR-23a-3p, and miR-451a) were significantly increased in the EDTA plasma. Moreover, we observed a time-dependent increase in plasma microRNAs that was most pronounced in the EDTA blood stored at room temperature for 24 h. Furthermore, significant correlations between microRNA levels and plasma concentrations of platelet-stored molecules pointed towards in vitro platelet activation. Therefore, we strongly recommend to (i) use CTAD as an anticoagulant, (ii) process blood samples as quickly as possible, and (iii) store blood samples at 4 °C whenever immediate plasma preparation is not feasible to generate reliable data on blood-derived microRNA signatures.
Project description:Castration-resistant prostate cancer (CRPC) is the major cause of death from prostate cancer. Biomarkers to improve early detection and prediction of CRPC especially using non-invasive liquid biopsies could improve outcomes. Therefore, we investigated the plasma exosomal miRNAs associated with CRPC and their potential for development into non-invasive early detection biomarkers for resistance to treatment. RNA-sequencing, which generated approximately five million reads per patient, was performed to identify differentially expressed plasma exosomal miRNAs in 24 treatment-naive prostate cancer and 24 CRPC patients. RT-qPCR was used to confirm the differential expressions of six exosomal miRNAs, miR-423-3p, miR-320a, miR-99a-5p, miR-320d, miR-320b, and miR-150-5p (<i>p</i> = 7.3 × 10<sup>-8</sup>, 0.0020, 0.018, 0.0028, 0.0013, and 0.0058, respectively) firstly in a validation cohort of 108 treatment-naive prostate cancer and 42 CRPC patients. The most significant differentially expressed miRNA, miR-423-3p, was shown to be associated with CRPC with area under the ROC curve (AUC) = 0.784. Combining miR-423-3p with prostate-specific antigen (PSA) enhanced the prediction of CRPC (AUC = 0.908). A separate research center validation with 30 treatment-naive and 30 CRPC patients also confirmed the differential expression of miR-423-3p (<i>p</i> = 0.016). Finally, plasma exosomal miR-423-3p expression in CRPC patients was compared to 36 non-CRPC patients under androgen depletion therapy, which showed significantly higher expression in CRPC than treated non-CRPC patients (<i>p</i> < 0.0001) with AUC = 0.879 to predict CRPC with no difference between treatment-naive and treated non-CRPC patients. Therefore, our findings demonstrate that a number of plasma exosomal miRNAs are associated with CRPC and miR-423-3p may serve as a biomarker for early detection/prediction of castration-resistance.
Project description:The potential of extracellular circulating microRNAs (miRNAs) as non-invasive biomarkers of atrial fibrillation (AF) has been confirmed by a number of recent studies. However, the current data for some miRNAs are controversial and inconsistent, probably due to pre-analytical and methodological differences. In this work, we attempted to fulfill the basic pre-analytical requirements provided for circulating miRNA studies for application to paroxysmal atrial fibrillation (PAF) research. We used quantitative PCR (qPCR) to determine the relative plasma levels of circulating miRNAs expressed in the heart or associated with atrial remodeling or fibrillation with reported altered plasma/serum levels in AF: miR-146a-5p, miR-150-5p, miR-19a-3p, miR-21-5p, miR-29b-3p, miR-320a-3p, miR-328-3p, miR-375-3p, and miR-409-3p. First, in a cohort of 90 adult outpatient clinic patients, we found that the plasma level of miR-320a-3p was elevated in PAF patients compared to healthy controls and hypertensive patients without AF. We further analyzed the impact of medication therapies on miRNA relative levels and found elevated miR-320a-3p levels in patients receiving angiotensin-converting-enzyme inhibitors (ACEI) therapy. Additionally, we found that miR-320a-3p, miR-21-5p, and miR-146a-5p plasma levels positively correlated with the CHA2DS2-Vasc score and were elevated in subjects with CHA2DS2-Vasc ? 2. Our results indicate that, amongst the analyzed miRNAs, miR-320a-3p may be considered as a potential PAF circulating plasma biomarker, leading to speculation as to whether this miRNA is a marker of platelet state change due to ACEI therapy.
Project description:Coronary artery disease (CAD) and acute myocardial infarction (AMI) are the leading causes of death worldwide. Since only a subset of CAD patients develops myocardial infarction, it is likely that unique factors predispose to AMI. Circulating microRNAs represent diagnostic powerful biomarkers for detection of heart injuries and patients' risk stratification. Using an array-based approach, the expression of 84 circulating miRNAs was analyzed in plasma of pooled stable CAD patients (CAD; n = 5) and unstable CAD patients (AMI_T0; n = 5) enrolled within 24 hours from an AMI event. The array experiments showed 27 miRNAs differentially expressed with a two-fold up- or down-regulation (10 up- and 17 down-regulated miRNAs). Among them, miR-423-5p dis-regulation was confirmed in a larger case study (n = 99). Circulating miR-423-5p resulted to be significantly down-regulated within 24 hours from the AMI event (FC = -2, p?0.05). Interestingly, miR-423-5p expression resulted to be increased (FC = +2; p?0.005) in a subgroup of the same AMI patients (AMI_T1; n = 11) analyzed after 6 months from the acute event. We extended miR-423-5p expression study on PBMCs (peripheral blood mononuclear cells), confirming also in this tissue its up-regulation at 6 months post-AMI. Receiver operating characteristic analyses (ROC) were performed to detect the power of miR-423-5p to discriminate stable and unstable CAD. In plasma, miR-423-5p expression accurately distinguishes stable and unstable CAD patients (AUC = 0.7143, p?0.005). Interestingly, the highest discriminatory value (AUC = 0.8529 p?0.0005) was identified in blood cells, where miR-423-5p expression is able to differentiate unstable CAD patients during an acute event (AMI_T0) from those at six months post-AMI (AMI_T1). Furthermore, cellular miR-423-5p may discriminate also stable CAD patients from unstable CAD patients after six months post-AMI (AUC = 0.7355 p?0.05). The results of this pilot-study suggest that miR-423-5p expression level both in plasma and blood cells, could represent a new promising biomarker for risk stratification of CAD patients.
Project description:Immune cell subsets and microRNAs have been independently proposed as type 1 diabetes (T1D) diagnostic and/or prognostic biomarkers. Here, we aimed to analyze the relationships between peripheral blood circulating immune cell subsets, plasmatic microRNAs, and T1D. Blood samples were obtained from both children with T1D at diagnosis and age-sex matched healthy controls. Then, immunophenotype assessed by flow cytometry was coupled with the quantification of 60 plasmatic microRNAs by quantitative RT-PCR. The associations between immune cell frequency, plasmatic microRNAs, and the parameters of pancreatic loss, glycemic control, and diabetic ketoacidosis were assessed by logistic regression models and correlation analyses. We found that the increase in specific plasmatic microRNAs was associated with T1D disease onset (let-7c-5p, let-7d-5p, let-7f-5p, let-7i-5p, miR-146a-5p, miR-423-3p, and miR-423-5p), serum C-peptide concentration (miR-142-5p and miR-29c-3p), glycated hemoglobin (miR-26a-5p and miR-223-3p) and the presence of ketoacidosis (miR-29c-3p) more strongly than the evaluated immune cell subset frequency. Some of these plasmatic microRNAs were shown to positively correlate with numbers of blood circulating B lymphocytes (miR-142-5p) and CD4+CD45RO+ (miR-146a-5p and miR-223-3p) and CD4+CD25+ cells (miR-423-3p and miR-223-3p) in children with T1D but not in healthy controls, suggesting a disease-specific microRNA association with immune dysregulation in T1D. In conclusion, our results suggest that, while blood co-circulating extracellular microRNAs and immune cell subsets may be biologically linked, microRNAs may better provide powerful information about T1D onset and severity.
Project description:Background:A reliable noninvasive biomarker is not yet available for endometriosis diagnosis. Novel biomarkers for the diagnosis of endometriosis are urgently needed. The molecular constituents of exosomes, especially exosomal microRNAs (miRNAs), have considerable potential as novel biomarkers for clinical diagnosis. This study is aimed at exploring aberrant exosomal miRNA profiles by using miRNA microarray and at providing more accurate molecular biomarkers of endometriosis. Methods:Exosomes were isolated from the serum of patients with endometriosis and negative controls and identified by electron microscopy, nanoparticle tracking analysis, and Western blot. Exosomal miRNAs were profiled by miRNA microarrays. The expression of selective serum exosomal miRNA was validated by qRT-PCR. Receiver operating characteristic (ROC) curves were established to explore the diagnostic value of selective miRNAs. Finally, GO annotation and KEGG pathway enrichment analyses were used to display possible functions associated with the two miRNAs. Results:A total of 24 miRNAs showed differential levels of enrichment with P < 0.05 and |log2?fold?change| > 1 by miRNA microarrays. Among the six selective miRNAs (i.e., miR-134-5p, miR-197-5p, miR-22-3p, miR-320a, miR-494-3p, and miR-939-5p), qRT-PCR analysis revealed that miR-22-3p and miR-320a were significantly upregulated in serum exosomes from patients with endometriosis compared with negative individuals. ROC curve revealed that the serum exosomal miR-22-3p and miR-320a yielded the area under the curve values of 0.855 and 0.827, respectively. Conclusion:Our results demonstrated that exosomal miR-22-3p and miR-320a were significantly increased in the sera of patients with endometriosis. The two miRNAs may be useful potential biomarkers for endometriosis diagnosis.
Project description:Cardiogenic shock (CS) is a life-threatening emergency. New biomarkers are needed in order to detect patients at greater risk of adverse outcome. Our aim was to assess the characteristics of miR-21-5p, miR-122-5p, and miR-320a-3p in CS and evaluate the value of their expression levels in risk prediction. Circulating levels of miR-21-5p, miR-122-5p, and miR-320a-3p were measured from serial plasma samples of 179 patients during the first 5-10 days after detection of CS, derived from the CardShock study. Acute coronary syndrome was the most common cause (80%) of CS. Baseline (0 h) levels of miR-21-5p, miR-122-5p, and miR-320a-3p were all significantly elevated in nonsurvivors compared to survivors (p < 0.05 for all). Above median levels at 0h of each miRNA were each significantly associated with higher lactate and alanine aminotransferase levels and decreased glomerular filtration rates. After adjusting the multivariate regression analysis with established CS risk factors, miR-21-5p and miR-320a-3p levels above median at 0 h were independently associated with 90-day all-cause mortality (adjusted hazard ratio 1.8 (95% confidence interval 1.1-3.0), p = 0.018; adjusted hazard ratio 1.9 (95% confidence interval 1.2-3.2), p = 0.009, respectively). In conclusion, circulating plasma levels of miR-21-5p, miR-122-5p, and miR-320a-3p at baseline were all elevated in nonsurvivors of CS and associated with markers of hypoperfusion. Above median levels of miR-21-5p and miR-320a-3p at baseline appear to independently predict 90-day all-cause mortality. This indicates the potential of miRNAs as biomarkers for risk assessment in cardiogenic shock.
Project description:The aims of this study were to compare microRNA (miR) expression between individuals with and without insulin resistance and to determine whether miRs predict response to thiazolidinedione treatment.In a sample of 93 healthy adults, insulin resistance was defined as steady state plasma glucose (SSPG)?180 mg/dL and insulin sensitive as <120 mg/dL. Response to thiazolidinedione therapy was defined as ?10% decrease in SSPG. We selected a panel of microRNAs based on prior evidence for a role in insulin or glucose metabolism. Fold change and Wilcoxon rank sum tests were calculated for the 25 miRs measured.At baseline, 81% (n=75) of participants were insulin resistant. Five miRs were differentially expressed between the insulin resistant and sensitive groups: miR-193b (1.45 fold change (FC)), miR-22-3p (1.15 FC), miR-320a (1.36 FC), miR-375 (0.59 FC), and miR-486 (1.21 FC) (all p<0.05). In the subset who were insulin resistant at baseline and received thiazolidinediones (n=47), 77% (n=36) showed improved insulin sensitivity. Six miRs were differentially expressed between responders compared to non-responders: miR-20b-5p (1.20 FC), miR-21-5p, (0.92 FC), miR-214-3p (1.13 FC), miR-22-3p (1.14 FC), miR-320a (0.98 FC), and miR-486-5p (1.25 FC) (all p<0.05).This study is the first to report miRs associated with response to a pharmacologic intervention for insulin resistance. MiR-320a and miR-486-5p identified responders to thiazolidinedione therapy among the insulin resistant group.
Project description:Cell-free microRNAs have been reported as biomarkers for several diseases. For testicular germ cell tumors (GCT), circulating microRNAs 371a-3p and 372-3p in serum and plasma have been proposed as biomarkers for diagnostic and disease monitoring purposes. The most widely used method for quantification of specific microRNAs in serum and plasma is reverse transcriptase real-time quantitative PCR (RT-qPCR) by the comparative Ct-method. In this method one or several reference genes or reference microRNAs are needed in order to normalize and calculate the relative microRNA levels across samples. One of the pitfalls in analysis of microRNAs from serum and plasma is the release of microRNAs from blood cells during hemolysis. This is an important issue because varying degrees of hemolysis are not uncommon in routine blood sampling. Thus, hemolysis must be taken into consideration when working with circulating microRNAs from blood. miR-93-5p, miR-30b-5p, and miR-20a-5p have been reported as reference microRNA in analysis of the miR-371a-373 cluster. We here show how these three microRNAs are influenced by hemolysis. We also propose a new reference microRNA, miR-191-5p, which is relatively stable in serum samples with mild hemolysis. In addition, we show how hemolysis can have effect on the reported microRNA levels in patient samples when these reference microRNAs are used in samples with varying levels of hemolysis.