Circulating Micro RNAs Research Articles

MicroRNA of extracellular vesicles as biomarkers and regulators of pathological and physiological processes

Extracellular vesicles (EV) are currently considered potential biomarkers of diseases, as well as “biological constructs” for targeted drug delivery. Extracellular vesicles represent a heterogeneous population of membrane vesicles formed by various cell populations, including immune cells. At present time, EV are divided into exosomes, microvesicles, and apoptotic bodies, depending on their size and formation manner. EV have been found in various human biological fluids. Therefore, the possibility of their usage as diagnostic biomarkers is under discussion. The vesicles have a diverse internal composition and express a wide repertoire of receptors on their surface, thus allowing them to participate in different intercellular communications by transferring to the cells various molecules, including genetic material. E.g., microRNAs transmitted via extracellular vesicles are evolutionarily conserved non-coding RNA molecules 18-25 nucleotides long. Their main function is to regulate gene expression at the post-transcriptional level. MicroRNAs are synthesized by different cell types. However, some microRNAs are found ubiquitously, whereas others are present only in certain types of tissues. MicroRNAs are found both inside, and outside the cells (extracellular or circulating microRNAs). The microRNAs are resistant to RNases and stable in the extracellular environment, due to their secretion as protein complexes, or as part of extracellular vesicles. The variable microRNA profile in extracellular vesicles depends on the physiological conditions and presence of various pathological disorders. Multiple studies show that microRNAs can determine the functional activity of extracellular vesicles, e.g., therapeutic usage of microRNAs carried by EV as well as diagnostic applications in various pathologies. This review considers distinct populations of extracellular vesicles and their main properties, describes the characteristics of intra- and extracellular (circulating) microRNAs, mechanisms of their biosynthesis, and techniques for detection and assessing contents of microRNAs. The review describes microRNAs as a component of exosomes and microvesicles formed by various cells, including cells of the immune system in the course of physiological and pathological processes, with respect to functions of these microRNAs as well as their diagnostic and therapeutic potential.

Circulatory miR-411-5p as a Novel Prognostic Biomarker for Major Adverse Cardiovascular Events in Patients with Atrial Fibrillation.

The risk stratification of patients with atrial fibrillation (AF) for subsequent cardiovascular events could help in guiding prevention strategies. In this study, we aimed at investigating circulating microRNAs as prognostic biomarkers for major adverse cardiovascular events (MACE) in AF patients. We conducted a three-stage nested case-control study within the framework of a prospective registry, including 347 AF patients. First, total small RNA-sequencing was performed in 26 patients (13 cases with MACE) and the differential expression of microRNAs was analyzed. Seven candidate microRNAs with promising results in a subgroup analysis on cardiovascular death were selected and measured via using RT-qPCR in 97 patients (42 cases with cardiovascular death). To further validate our findings and investigate broader clinical applicability, we analyzed the same microRNAs in a subsequent nested case-control study of 102 patients (37 cases with early MACE) by using Cox regression. In the microRNA discovery cohort (n = 26), we detected 184 well-expressed microRNAs in circulation without overt differential expression between the cases and controls. A subgroup analysis on cardiovascular death revealed 26 microRNAs that were differentially expressed at a significance level < 0.05 (three of which with an FDR-adjusted p-value <0.05). We, therefore, proceeded with a nested case-control approach (n = 97) focusing on patients with cardiovascular death and selected, in total, seven microRNAs for further RT-qPCR analysis. One microRNA, miR-411-5p, was significantly associated with cardiovascular death (adjusted HR (95% CI): 1.95 (1.04-3.67)). Further validation (n = 102) in patients who developed early MACE showed similar results (adjusted HR (95% CI) 2.35 (1.17-4.73)). In conclusion, circulating miR-411-5p could be a valuable prognostic biomarker for MACE in AF patients.

Circulating microRNAs in gallbladder cancer: Is serum assay of diagnostic value?

The microRNAs (miRNAs) in circulation could serve as biomarkers for cancer detection. Gallbladder carcinoma (GBC) is mostly asymptomatic; therefore, using microRNAs (miRNAs) as an early diagnostic biomarker could be a valuable tool. We aimed to identify the tumor-associated miR-1, miR130, miR-146, miR-182, and miR-21expression in serum as a biomarker for early detection of GBC and identify their possible diagnostic role. The study group comprised of paired serum and tissue samples from 34 GBC, 19 cholecystitis (CC), 21 normal controls (uninflamed gall bladder), and additional 29 serum-only samples of GBC. Total RNA was isolated using a commercially available RNA isolation kit (Applied Biosystem, USA) and reverse transcribed using Advanced Taqman MicroRNA reverse transcription kit. The relative expression of miRNAs was analyzed using Quantitative real-time polymerase chain reaction. The diagnostic potential of these miRNAs was assessed by ROC analysis. In paired samples, the trend towards up and down regulation for miR-182, miR-21, miR-1, miR-130, and miR-146 was similar in both tissue and sera of GBC. The expression pattern of serum miR-1, miR130, and miR-146 gradually decreased from normal control (NC) to CC to GBC, while miR-21 and miR-182 gradually increased from NC to CC to GBC. The miR-1, miR-121, miR-182, and miR-146 significantly differed between CC vs. early stage and early stage vs. NC. Among these miRNAs, the sensitivity of miR-1 (85.71 %) was the highest, and the specificity of miR-21 was the highest (92.73 %). The combined sensitivity for miRNAs ranged from 73.13 % (CI: 60.90-83.24 %) to 98.63 % (CI: 89.0-99.61 %); however, the specificity was lower. In stage I&II vs. III&IV discrimination, the diagnostic sensitivity of miR-1 was highest (89.36 %, CI: 76.90-96.45). The two miRNAs, in combination, increase the diagnostic sensitivity. Circulating serum miRNAs may provide a new approach for clinical application. Panels of specific circulating miRNA, which require further validation, could be potential non-invasive diagnostic biomarkers for GBC in combination with abnormal radio diagnostic scans.

Plasma circulating micro-RNAs associated with alexithymia reflect a high overlap on neuropsychiatric outcomes

BackgroundAlexithymia (“no word for feelings”) is a personality feature that is common in patients with psychiatric disorders. However, little is known about biological causes and mechanism behind. Research so far focusses on genetic risk variants primary associated with depression, but analyses on epigenetic mechanisms are still missing. MethodsIn a sample of n = 813 subjects from the “Study of Health in Pomerania” we analyzed the association between alexithymia and plasma circulating micro RNAs (miRNA). Significant miRNAs were compared to associations regarding depression and pathway analyses were performed. ResultsTwo miRNAs were significantly associated with at least one of the alexithymia scores (hsa-miR-324-3p, hsa-miR-33a-5p) and three miRNAs additionally revealed a strong association pattern to alexithymia (hsa-miR-106b-5p, hsa-miR-484, hsa-miR-25-3p). None of these miRNAs was significantly associated with depressive symptoms in our sample. Literature research showed that all of the miRNAs have been found in association with several neuropsychiatric phenotypes. LimitationsMain limitations are the lack of a replication sample as well as the limited number of miRNAs analyzed. ConclusionsThis is the first analysis investigating the association between miRNAs and alexithymia. Results indicate that miRNAs are not specific for one psychiatric disorder but may influence biological mechanisms that are involved in various psychiatric conditions which might be relevant for future treatment options and improve the biological understanding of psychiatric conditions.

Circulating MicroRNAs Highly Correlate to Expression of Cartilage Genes Potentially Reflecting OA Susceptibility-Towards Identification of Applicable Early OA Biomarkers.

Objective: To identify and validate circulating micro RNAs (miRNAs) that mark gene expression changes in articular cartilage early in osteoarthritis (OA) pathophysiology process. Methods: Within the ongoing RAAK study, human preserved OA cartilage and plasma (N = 22 paired samples) was collected for RNA sequencing (respectively mRNA and miRNA). Spearman correlation was determined for 114 cartilage genes consistently and significantly differentially expressed early in osteoarthritis and 384 plasma miRNAs. Subsequently, the minimal number of circulating miRNAs serving to discriminate between progressors and non-progressors was assessed by regression analysis and area under receiver operating curves (AUC) was calculated with progression data and plasma miRNA sequencing from the GARP study (N = 71). Results: We identified strong correlations (ρ ≥ |0.7|) among expression levels of 34 unique plasma miRNAs and 21 genes, including 4 genes that correlated with multiple miRNAs. The strongest correlation was between let-7d-5p and EGFLAM (ρ = −0.75, P = 6.9 × 10−5). Regression analysis of the 34 miRNAs resulted in a set of 7 miRNAs that, when applied to the GARP study, demonstrated clinically relevant predictive value with AUC > 0.8 for OA progression over 2 years and near-clinical value for progression over 5 years- (AUC = 0.8). Conclusions: We show that plasma miRNAs levels reflect gene expression levels in cartilage and can be exploited to represent ongoing pathophysiological processes in articular cartilage. We advocate that identified signature of 7 plasma miRNAs can contribute to direct further studies toward early biomarkers predictive for progression of osteoarthritis over 2 and 5 years.

Modulations of obesity-related microRNAs after exercise intervention: a systematic review and bioinformatics analysis.

Obesity is one of the prevalent health-threatening conditions; however, it is preventable by lifestyle interventions such as exercise. The molecular mechanisms underlying physiological adaptation to physical activity are not fully understood. It has been documented that both intracellular and extracellular (circulating) microRNAs (miRNAs) are involved in both obesogenic and exercise adaptation mechanisms. We aimed to conduct a systematic review of publications that examined the effect of exercise on the expression of miRNAs in individuals with obesity. In addition, bioinformatics analysis was performed on most repetitive miRNAs. PubMed, Scopus, and Google Scholar were searched with relevant keywords. We only included studies that utilized exercise as a modality for the health management of human subjects with obesity to evaluate the changes in expression of obesity-related miRNAs. Through checking of 211 retrieved articles, we reached 12 eligible studies. Some studies reported a statistically significant correlation between the change of miRNAs and clinical parameters such as body mass index and fasting glucose. In silico analysis of most repetitive miRNAs i.e. miR-126, miR-21, miR-146a, miR-221, and miR-223 resulted in the molecular signaling pathways that potentially involve in cellular adaption to exercise in people with obesity. miRNAs partake in health-related benefits of physical activity on obesity-associated cellular and molecular phenomena. However, our understanding of the exact mechanism is still in its infancy. Consistently, the clinicians waiting for the result of more integrated experiments to develop a miRNAs panel as a predictive biomarker of exercise in patients with obesity.

Circulating MicroRNAs as Diagnostic Biomarkers for Motor Neuron Disease.

Motor neuron disease (MND) is a kind of neurodegenerative disease that selectively invades spinal cord anterior horn cells, brainstem motor neurons, cortical pyramidal cells and the pyramidal tract. The main clinical features are the symptoms and signs of impaired upper and lower motor neurons, manifested as muscle weakness, atrophy and pyramidal tract signs. Histopathology has shown the disappearance of pyramidal cells in the motor cortex, loss of motor neurons in the anterior horn of the spinal cord and brainstem, and degeneration of the corticospinal tract. Due to the lack of effective treatment methods, the prognosis is generally poor, so it is of great significance to confirm the diagnosis early by various means. However, the current diagnosis of MND mainly relies on the combination of clinical manifestations and neurophysiological examinations, lacking effective means of early diagnosis. Circulating microRNA (CmiRNA) is a kind of stable miRNA molecule in serum, plasma and other body fluids, which has the characteristics of distinct differential expression, sensitive detection and convenient sample collection. As a possible new biomarker of MND, CmiRNA can not only reveal the pathophysiological process of MND, but also monitor disease progression and response to drug therapy. With the development of miRNA detection technology, more and more CmiRNAs as biomarkers with potential diagnostic value have been investigated. In this review, we explored the possibility of circulating samples as different sources of biomarkers for the diagnosis of MND, analyzing the progress of CmiRNA detection techniques, and presenting potential diagnostic MND biomarkers that have been reported.

Expression profile of maternal circulating microRNAs as non-invasive biomarkers for prenatal diagnosis of congenital heart defects

ObjectiveThe discovery of cell free fetal microRNAs (miRNAs) in maternal circulation has opened up novel probabilities for non-invasive prenatal diagnosis. This study aims to investigate circulating miRNAs as potential biomarkers in the serum of pregnant women with congenital heart defect (CHD) fetuses. MethodA total of 110 pregnant women including 50 CHD cases and 60 healthy controls were included in this study. miRNA microarray followed by real-time PCR was used to explore miRNA expression. Receiver operating characteristic (ROC) curves were calculated to assess the diagnostic capability of miRNAs for fetal CHDs. Results38 Serum miRNAs were revealed to be differentially expressed in the CHD group as compared to control group via microarray. Among these, nine down-regulated and three up-regulated miRNAs were validated by real-time PCR. Ten of these miRNAs were rapidly reduced in the normal maternal serum after delivery as compared to before delivery. In particular, we identified a biomarker panel consisting of four miRNAs (miR-142-5p, miR-1275, miR-4666a-3p and miR-3664-3p) capable of distinguishing CHDs from controls (area under the ROC curve (AUC), 0.920; p < 0.0001). ConclusionThe discovery of these dysregulated pregnancy-associated miRNAs in maternal serum may be potential biomarkers for non-invasive prenatal diagnosis of fetal CHDs.

Circulating micro-RNAs as biomarkers of coronary artery disease: is it ready for primetime or still a work in progress?

MicroRNAs (miRs) are small non-coding RNAs that play a significant role in epigenetic regulation of gene expression (1,2). Several miRs have been established to play crucial roles in a spectrum of different cardiovascular diseases (3,4). Since the initial identification of detectable levels of circulating miRs in plasma (5-7), circulating miRs have become the spotlight of investigation as potential biomarkers for several diseases. Micro-RNAs in circulation have two important features of ideal biomarkers: they are remarkably stable and safe from degradation by RNases, and they are easily measured in the circulation using sensitive and specific quantitative PCR techniques. Thus, circulating miRs can be linked to specific disease processes or to specific tissue and/or cell types.

Performance of Serum microRNAs -122, -192 and -21 as Biomarkers in Patients with Non-Alcoholic Steatohepatitis.

ObjectivesLiver biopsies are the current gold standard in non-alcoholic steatohepatitis (NASH) diagnosis. Their invasive nature, however, still carries an increased risk for patients’ health. The development of non-invasive diagnostic tools to differentiate between bland steatosis (NAFL) and NASH remains crucial. The aim of this study is the evaluation of investigated circulating microRNAs in combination with new targets in order to optimize the discrimination of NASH patients by non-invasive serum biomarkers.MethodsSerum profiles of four microRNAs were evaluated in two cohorts consisting of 137 NAFLD patients and 61 healthy controls. In a binary logistic regression model microRNAs of relevance were detected. Correlation of microRNA appearance with known biomarkers like ALT and CK18-Asp396 was evaluated. A simplified scoring model was developed, combining the levels of microRNA in circulation and CK18-Asp396 fragments. Receiver operating characteristics were used to evaluate the potential of discriminating NASH.ResultsThe new finding of our study is the different profile of circulating miR-21 in NASH patients (p<0.0001). Also, it validates recently published results of miR-122 and miR-192 to be differentially regulated in NAFL and NASH. Combined microRNA expression profiles with CK18-Asp396 fragment level scoring model had a higher potential of NASH prediction compared to other risk biomarkers (AUROC = 0.83, 95% CI = 0.754–0.908; p<0.001). Evaluation of score model for NAFL (Score = 0) and NASH (Score = 4) had shown high rates of sensitivity (91%) and specificity (83%).ConclusionsOur study defines candidates for a combined model of miRNAs and CK18-Asp396 levels relevant as a promising expansion for diagnosis and in turn treatment of NASH.

Circulating microRNAs as prognostic therapy biomarkers in head and neck cancer patients.

Background:The prediction of therapy response in head and neck squamous cell cancer (HNSCC) requires biomarkers, which are also a prerequisite for personalised therapy concepts. The current study aimed to identify therapy-responsive microRNAs (miRNAs) in the circulation that can serve as minimally invasive prognostic markers for HNSCC patients undergoing radiotherapy.Methods:We screened plasma miRNAs in a discovery cohort of HNSCC patients before therapy and after treatment. We further compared the plasma miRNAs of the patients to age- and sex-matched healthy controls. All miRNAs identified as biomarker candidates were then confirmed in an independent validation cohort of HNSCC patients and tested for correlation with the clinical outcome.Results:We identified a signature of eight plasma miRNAs that differentiated significantly (P=0.003) between HNSCC patients and healthy donors. MiR-186-5p demonstrated the highest sensitivity and specificity to classify HNSCC patients and healthy individuals. All therapy-responsive and patient-specific miRNAs in plasma were also detectable in tumour tissues derived from the same patients. High expression of miR-142-3p, miR-186-5p, miR-195-5p, miR-374b-5p and miR-574-3p in the plasma correlated with worse prognosis.Conclusions:Circulating miR-142-3p, miR-186-5p, miR-195-5p, miR-374b-5p and miR-574-3p represent the most promising markers for prognosis and therapy monitoring in the plasma of HNSCC patients. We found strong evidence that the circulating therapy-responsive miRNAs are tumour related and were able to validate them in an independent cohort of HNSCC patients.

Abstract 355: Dyslipidemia, Obesity, Insulin Resistance and Atherosclerosis in LDL-Receptor Deficient Rats

The LDL-receptor (LDLR) plays a major role in removing circulating LDL. Deletion of the receptor in mice results in profound dyslipidemia and atherosclerotic lesion formation. We established a new LDLR deficient (KO) rat model of atherosclerosis. When maintained on normal chow (NC), the KO rats displayed a 2.5-fold increase in plasma total cholesterol and 1.5 to 2.0- fold increase in triglycerides levels than corresponding WT rats. Lipidomics analysis of the plasma showed that deficiency of LDLR significantly (P&lt;0.05) affected the abundance of 65 lipids. On normal chow, KO rats gained more weight and were more glucose intolerant than WT rats. Plasma leptin levels were higher and adiponectin levels were lower in KO than WT rats. Untargeted metabolomics analysis of polar extracts of plasma revealed that deficiency of LDLR differentially regulated the abundance of 20 metabolites (P&lt;0.05). Measurement of circulating micro RNAs showed differential regulation of 35 micro RNAs, several of which are associated with obesity, insulin resistance and dyslipidemia. On Western diet (WD), the KO rats displayed exaggerated obesity and age-dependent increase in glucose intolerance. Their plasma levels of adiponectin and leptin were lower in comparison with WT rats. Lipidomics and metabolomics analyses of plasma showed differential regulation of 53 lipids (P&lt;0.05) and 9 polar metabolites (P&lt;0.05) as compared with WT rats maintained on WD. No appreciable aortic lesions were observed in KO rats fed NC for 64 weeks or WD for 16 weeks; however after 34-52 weeks of Western diet, the KO rats developed exuberant atherosclerotic lesions in the aortic arch and throughout the abdominal aorta. Several microRNAs associated with atherosclerosis, insulin resistance and obesity were also differentially regulated in the KO rats maintained on WD. These data suggest that LDLR-KO rat may be a useful model for studying obesity, insulin resistance, and atherosclerosis; and a panel of signature plasma lipids, polar metabolites and circulating microRNAs may serve as biomarkers of atherosclerosis and its risk factors.

Abundant circulating microRNAs in breast cancer patients fluctuate considerably during neoadjuvant chemotherapy.

Previous studies have revealed the aberrant expression of a number of microRNAs (miRNA/miRs) in the blood circulation of patients with breast cancer (BC). The aim of the present study was to assess the effect of neoadjuvant chemotherapy on the levels of a panel of BC-associated miRNAs, which are at relatively low (let-7, miR-10b, miR-34, miR-155, miR-200c and miR-205) or abundant (miR-21, miR-195 and miR-221) levels in the circulation. Patients with primary operable or locally advanced BC were enrolled in the study. The plasma levels of the miRNAs at baseline and at the fourth cycle of treatment were compared. Patients with stage II disease exhibited higher basal miRNAs levels than those with higher stages. The difference was most evident for miR-155 and miR-21 (P=0.05). From the initial to the fourth cycle of chemotherapy, the miRNA levels changed substantially. In samples in which the miRNA levels generally declined, a marked decrease (≤15,500-fold) was evident for the abundant miRNAs. Notably, the occurrence of a decrease in miRNA levels was more frequent in patients with smaller tumor sizes (P<0.05 for miR-21 and miR-195). This proof-of-concept study provides evidence that highly expressed miRNAs are affected most frequently by chemotherapy, particularly in patients with early stage tumors. This information may be valuable in assessing the response of the patients to therapy.

Searching for circulating microRNAs in genitourinary tumors.

468 Background: Detection of circulating tumor cells (CTC) may provide diagnostic and prognostic information in genitourinary tumors (GT). The aim of this work was identify aberrantly expressed miRNAs potentially useful for CTC detection in blood samples from patients with GT to assess their potential clinical significance, and to gain a greater understanding of the mechanisms driving tumor progression. Methods: We examined blood levels of 92 microRNAs in 113 metastatic patients: prostate (mP), renal cell carcinoma (mRCC), bladder tumors (mB) and healthy volunteers (HV) (N=18) using SYBR-green-based microRNA RT-qPCR arrays (Exiqon). Array design was made by literature review and bioinformatics approach using free databases. Relative Expression Software Tool (REST) and SPPS 21.0 were used to data processing and analysis. Results: microRNAs differential expressed between tumors and HV (only up-regulated miRNAs are shown in Table). Conclusions: microRNAs in the circulation are relatively stable, very accessible, low invasive and easily testable biomarkers. These results suggest that our bioinformatic approach is an useful high-throughput method to identified tumors-associated miRNAs. The selected miRNAs should be further evaluated for their potential as markers for CTC detection, prognosis and therapeutic outcome. Further studies of precision and sensitivity on these markers in this GT population will clarify its role as novel stable blood-based markers. [Table: see text]

MicroRNAs in Circulation Are Altered in Response to Influenza A Virus Infection in Humans

Changes in microRNA expression have been detected in vitro in influenza infected cells, yet little is known about them in patients. microRNA profiling was performed on whole blood of H1N1 patients to identify signature microRNAs to better understand the gene regulation involved and possibly improve diagnosis. Total RNA extracted from blood samples of influenza infected patients and healthy controls were subjected to microRNA microarray. Expression profiles of circulating microRNAs were altered and distinctly different in influenza patients. Expression of highly dysregulated microRNAs were validated using quantitative PCR. Fourteen highly dysregulated miRNAs, identified from the blood of influenza infected patients, provided a clear distinction between infected and healthy individuals. Of these, expression of miR-1260, -26a, -335*, -576-3p, -628-3p and -664 were consistently dysregulated in both whole blood and H1N1 infected cells. Potential host and viral gene targets were identified and the impact of microRNA dysregulation on the host proteome was studied. Consequences of their altered expression were extrapolated to changes in the host proteome expression. These highly dysregulated microRNAs may have crucial roles in influenza pathogenesis and are potential biomarkers of influenza.

The Impact of Hemolysis on Cell-Free microRNA Biomarkers

Cell-free microRNAs in plasma and serum have become a promising source of biomarkers for various diseases. Despite rapid progress in this field, there remains a lack of consensus regarding optimal quantification methods, reference genes, and quality control of samples. Recent studies have shown that hemolysis occurring during blood collection has substantial impact on the microRNA content in plasma/serum. To date, the impact of hemolysis has only been investigated for a limited number of microRNAs, mainly the red blood cell (RBC)-enriched miRs-16 and -451. In contrast, the effect of hemolysis on other microRNAs – in particular those proposed as biomarkers – has not been addressed. In this study we profiled the microRNA content of hemolyzed and non-hemolyzed plasma as well as RBCs to obtain a profile of microRNAs in the circulation affected or unaffected by hemolysis. Profiling by TaqMan Array Microfluidic Cards was used to compare three pairs of hemolyzed and non-hemolyzed plasma (with varying degrees of hemolysis) and one RBC sample. A total of 136 microRNAs were detectable in at least two of the samples, and of those 15 were at least twofold elevated in all three hemolyzed samples. This number increased to 88 microRNAs for the sample with the highest level of hemolysis, with all of these also detected in the RBC profile. Thus these microRNAs represent a large proportion of detectable microRNAs and those most likely to be affected by hemolysis. Several of the hemolysis-susceptible microRNAs (e.g., miRs-21, -106a, -92a, -17, -16) have also been previously proposed as plasma/serum biomarkers of disease, highlighting the importance of rigorous quality control of plasma/serum samples used for measurement of circulating microRNAs. As low-level hemolysis is a frequent occurrence during plasma/serum collection it is critical that this is taken into account in the measurement of any candidate circulating microRNA.

Circulating MicroRNAs Are Not Eliminated by Hemodialysis

BackgroundCirculating microRNAs are stably detectable in serum/plasma and other body fluids. In patients with acute kidney injury on dialysis therapy changes of miRNA patterns had been detected. It remains unclear if and how the dialysis procedure itself affects circulating microRNA level.MethodsWe quantified miR-21 and miR-210 by quantitative RT-PCR in plasma of patients with acute kidney injury requiring dialysis and measured pre- and post-dialyser miRNA levels as well as their amount in the collected spent dialysate. Single treatments using the following filters were studied: F60 S (1.3 m2, Molecular Weight Cut Off (MWCO): 30 kDa, n = 8), AV 1000 S (1.8 m2, MWCO: 30 kDa, n = 6) and EMiC 2 (1.8 m2, MWCO: 40 kDa, n = 6).ResultsCirculating levels of miR-21 or -210 do not differ between pre- and post-dialyzer blood samples independently of the used filter surface and pore size: miR-21: F60S: p = 0.35, AV 1000 S p = 1.0, EMiC2 p = 1.0; miR-210: F60S: p = 0.91, AV 1000 S p = 0.09, EMiC2 p = 0.31. Correspondingly, only traces of both miRNAs could be found in the collected spent dialysate and ultrafiltrate.ConclusionsIn patients with acute kidney injury circulating microRNAs are not removed by dialysis. As only traces of miR-21 and -210 are detected in dialysate and ultrafiltrate, microRNAs in the circulation are likely to be transported by larger structures such as proteins and/or microvesicles. As miRNAs are not affected by dialysis they might be more robust biomarkers of acute kidney injury.

Abstract 4130: Bariatric surgery profoundly changes circulating MicroRNA profile in the rat

Abstract Obesity is a worldwide public health issue which can lead to cardiovascular disease, type 2 diabetes, and cancers. Bariatric surgery can provide sustained weight loss and a significant reduction of cancer risk. MicroRNAs are approximately 22 nucleotide long post transcription regulators. MicroRNAs are suggested to play important roles in obesity and metabolic syndrome related biological processes, but also are involved in different types of cancer. Recently microRNAs have been shown to be present in blood, and it is proposed that circulating microRNAs may be biomarker of tissue biochemistry and pathology. The aim of this study is to investigate if the circulation microRNA profile is changed by bariatric surgery and whether the altered microRNA expression could contribute to the long term reduced predisposition cancer risk associated with such surgery. Total plasma RNA from 53 days post bariatric surgery and sham operated rats were isolated and then examined using Taqman Rodent MicroRNA A+B Cards which have the capacity to evaluate expression levels of 373 rat microRNAs. Four different normalization methods (Global, cel-miR-39 spiked in, U6 endogenous and five endogenous microRNAs normalization) were tested. Five endogenous microRNAs normalization was found to be optimal and selected for all downstream analysis. Our preliminary data showed that 113 microRNAs were expressed in the plasma of bariatric surgical rats and 92 in sham operated rats. Furthermore, an OPLS-DA model clearly showed that bariatric surgery changed the circulating microRNA expression profile. Among nineteen of the most significantly changed microRNAs, nine of them are relevant to cancer. In summary, our data suggest that bariatric surgery fundamentally changed circulating microRNA expression. Understanding the significance of these changes in circulating microRNA may give clues to the mechanisms whereby bariatric surgery affords health benefits to individual undergoing such surgery, including a reduced predisposition to disease such as cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4130. doi:1538-7445.AM2012-4130

Microparticles: major transport vehicles for distinct microRNAs in circulation

AimsCirculating microRNAs (miRNAs) have attracted major interest as biomarkers for cardiovascular diseases. Since RNases are abundant in circulating blood, there needs to be a mechanism protecting miRNAs from degradation. We hypothesized that microparticles (MP) represent protective transport vehicles for miRNAs and that these are specifically packaged by their maternal cells.Methods and resultsConventional plasma preparations, such as the ones used for biomarker detection, are shown to contain substantial numbers of platelet-, leucocyte-, and endothelial cell-derived MP. To analyse the widest spectrum of miRNAs, Next Generation Sequencing was used to assess miRNA profiles of MP and their corresponding stimulated and non-stimulated cells of origin. THP-1 (monocytic origin) and human umbilical vein endothelial cell (HUVEC) MP were used for representing circulating MP at a high purity. miRNA profiles of MP differed significantly from those of stimulated and non-stimulated maternal THP-1 cells and HUVECs, respectively. Quantitative reverse transcription–polymerase chain reaction of miRNAs which have been associated with cardiovascular diseases also demonstrated significant differences in miRNA profiles between platelets and their MP. Notably, the main fraction of miRNA in plasma was localized in MP. Furthermore, miRNA profiles of MP differed significantly between patients with stable and unstable coronary artery disease.ConclusionCirculating MP represent transport vehicles for large numbers of specific miRNAs, which have been associated with cardiovascular diseases. miRNA profiles of MP are significantly different from their maternal cells, indicating an active mechanism of selective ‘packaging’ from cells into MP. These findings describe an interesting mechanism for transferring gene-regulatory function from MP-releasing cells to target cells via MP circulating in blood.