Circulating microRNAs as biomarkers of Chagas cardiomyopathy.

Background: Chagas cardiomyopathy (CHCM) is the most important clinical manifestation of Chagas disease. The analysis of cardiac miRNAs may contribute to predicting the progression to CHCM in Chagas indeterminate phase and/or to the differential diagnosis for cardiomyopathy. Methods: We carried out a case-control study to identify circulating miRNAs associated with CHCM. We assigned 104 participants to four groups: healthy controls (HC), Chagas non-cardiomyopathy controls, CHCM cases, and ischemic cardiomyopathy controls. We performed a clinical, echocardiographic, and laboratory evaluation and profiled circulating miRNA in the serum samples. Results: Differences between groups were observed in clinical variables and in the analysis of miRNAs. Compared to HC, CHCM participants had 4 over-expressed and 6 under-expressed miRNAs; miR-95-3p and miR-130b-3p were upregulated in CHCM compared with controls, Chagas non-cardiomyopathy and ischemic cardiomyopathy participants, suggesting that might be a hallmark of CHCM. Analysis of gene targets associated with cardiac injury yielded results of genes involved in arrhythmia generation, cardiomegaly, and hypertrophy. Conclusions: Our data suggest that the expression of circulating miRNAs identified by deep sequencing in CHCM could be associated with different cardiac phenotypes in CHCM subjects, compared with Chagas non-CHCM, ischemic cardiomyopathy controls, and healthy controls.

Circulating MicroRNAs as Novel Potential Diagnostic Biomarkers for Osteosarcoma: A Systematic Review.

Osteosarcoma (OS) is a fast-progressing bone tumor with high incidence in children and adolescents. The main diagnostic methods for OS are imaging exams and biopsies. In spite of the several resources available for detecting the disease, establishing an early diagnosis is still difficult, resulting in worse prognosis and lower survival rates for patients with OS. The identification of novel biomarkers would be helpful, and recently, circulating microRNAs (miRNAs) have been pointed to as possible non-invasive biomarkers. In order to assess the effectiveness of miRNA research, we performed a systematic review to assess the potential role of circulating miRNAs as biomarkers for OS diagnosis. We performed a search in various databases-PubMed, LILACS (Literatura Latino-americana e do Caribe em Ciências da Saúde), VHL (Virtual Health Library), Elsevier, Web of Science, Gale Academic One File-using the terms: "Circulating microRNAs" OR "plasma microRNAs" OR "serum microRNAs" OR "blood microRNAs" OR "cell-free microRNAs" OR "exosome microRNAs" OR "extracellular vesicles microRNAs" OR "liquid biopsy" AND "osteosarcoma" AND "diagnostic". We found 35 eligible studies that were independently identified and had had their quality assessed according to Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) guidelines. Despite the useful number of publications on this subject and the fact that several microRNAs showed excellent diagnostic performance for OS, the lack of consistency in results suggests that additional prospective studies are needed to confirm the role of circulating miRNAs as non-invasive biomarkers in OS.

Cigarette Smoking Alters the Expression of Circulating microRNAs and Its Potential Diagnostic Value in Female Lung Cancer Patients.

Cigarette smoking is a known risk factor for the development of lung cancer. We investigated whether circulating microRNA expression levels and their potential diagnostic value are affected by cigarette smoking in adenocarcinoma (AD) patients and healthy (H) participants. In total, 71 female AD patients and 91 H individuals were recruited, including 42 AD never-smokers (AD/CS-), 29 AD smokers (AD/CS+), 54 H never-smokers (H/CS-), and 37 H smokers (H/CS+). PCR array (754 microRNAs) and qPCR were performed on sera from the discovery and validation cohorts, respectively. The expression levels of miR-532-5p, miR-25-3p, and miR-133a-3p were significantly higher in adenocarcinoma patients than in healthy participants, independent of their smoking status. Multivariate analysis showed that levels of miR-133a-3p were independently associated with smoking. ROC analysis showed that only miR-532-5p discriminated AD patients from H controls (AUC: 0.745). However, when making comparisons according to cigarette smoking status, miR-532-5p discriminated AD/CS- patients from H/CS- controls with a higher AUC (AUC:0.762); miR-25-3p discriminated AD/CS+ patients from H/CS+ controls (AUC: 0.779), and miR-133a discriminated AD/CS+ patients from H/CS+ controls with the highest AUC of 0.935. Cancer and lung-cancer-enriched pathways were significantly associated with the three miRNAs; in addition, nicotinate/nicotinamide metabolism, inflammation, and pulmonary hypertension were associated with miR-133a-3p. Our findings highlight how cigarette smoking affects the reliable identification of circulating miRNAs as diagnostic biomarkers in lung cancer and suggest a smoking-dependent pathogenic role of miR-133a-3p in smokers.

MicroRNA-33b is a Potential Non-Invasive Biomarker for Response to Atorvastatin Treatment in Chilean Subjects With Hypercholesterolemia: A Pilot Study.

Evidence accumulated so far indicates that circulating levels of microRNAs (miRNAs) are associated with several pathologies. Therefore, differential expression of extracellular miRNAs exhibits promising potential for screening and diagnosis purposes. We evaluated plasma miRNAs in response to the lipid-lowering drug atorvastatin in patients with hypercholesterolemia (HC) and controls. METHODS: We selected miRNAs based on previous data reported by our group and also by employing bioinformatics tools to identify 10 miRNAs related to cholesterol metabolism and statin response genes. Following miRNA identification, we determined plasma levels of miRNA-17-5p, miRNA-30c-5p, miRNA-24-3p, miRNA-33a-5p, miRNA-33b-5p, miRNA-29a-3p, miRNA-29b-3p, miRNA-454-3p, miRNA-590-3p and miRNA-27a-3p in 20 HC patients before and after 1 month of 20 mg/day atorvastatin treatment, evaluating the same miRNA set in a group of 20 healthy subjects, and employing qRT-PCR to determine differential miRNAs expression. RESULTS: HC individuals showed significant overexpression of miRNA-30c-5p and miRNA-29b-3p vs. NL (p = 0.0008 and p = 0.0001, respectively). Once cholesterol-lowering treatment was concluded, HC individuals showed a substantial increase of three extracellular miRNAs (miRNA-24-3p, miRNA-590, and miRNA-33b-5p), the latter elevated more than 37-fold (p = 0.0082). CONCLUSION: Data suggest that circulating miRNA-30c-5p and miRNA-29b-3p are associated with hypercholesterolemia. Also, atorvastatin induces a strong elevation of miRNA-33b-5p levels in HC individuals, which could indicate an important function that this miRNA may exert upon atorvastatin therapy. Additional studies are needed to clarify the role of this particular miRNA in statin treatment.

Circulating MicroRNAs in Blood and Other Body Fluids as Biomarkers for Diagnosis, Prognosis, and Therapy Response in Lung Cancer.

The identification of circulating microRNAs (miRNAs) in peripheral blood and other body fluids has led to considerable research interest in investigating their potential clinical application as non-invasive biomarkers of cancer, including lung cancer, the deadliest malignancy worldwide. Several studies have found that alterations in the levels of miRNAs in circulation are able to discriminate lung cancer patients from healthy individuals (diagnosis) and are associated with patient outcome (prognosis) and treatment response (prediction). Increasing evidence indicates that circulating miRNAs may function as mediators of cell-to-cell communication, affecting biological processes associated with tumor initiation and progression. This review is focused on the most recent studies that provide evidence of the potential value of circulating miRNAs in blood and other body fluids as non-invasive biomarkers of lung cancer in terms of diagnosis, prognosis, and response to treatment. The status of their potential clinical application in lung cancer is also discussed, and relevant clinical trials were sought and are described. Because of the relevance of their biological characteristics and potential value as biomarkers, this review provides an overview of the canonical biogenesis, release mechanisms, and biological role of miRNAs in lung cancer.

Circulating microRNA Signature Associated to Interstitial Lung Abnormalities in Respiratory Asymptomatic Subjects.

Interstitial lung abnormalities (ILA) are observed in around 9% of older respiratory asymptomatic subjects, mainly smokers. Evidence suggests that ILA may precede the development of interstitial lung diseases and may evolve to progressive fibrosis. Identifying biomarkers of this subclinical status is relevant for early diagnosis and to predict outcome. We aimed to identify circulating microRNAs (miRNAs) associated to ILA in a cohort of respiratory asymptomatic subjects older than 60 years. We identified 81 subjects with ILA from our Lung-Aging Program in Mexico City (n = 826). We randomly selected 112 subjects without ILA (Ctrl) from the same cohort. Using polymerase chain reaction PCR-Array technology (24 ILA and 24 Ctrl, screening cohort) and reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) (57 ILA and 88 Ctr, independent validation cohort) we identified seven up-regulated miRNAs in serum of ILA compared to Ctrl (miR-193a-5p, p < 0.0001; miR-502-3p, p < 0.0001; miR-200c-3p, p = 0.003; miR-16-5p, p = 0.003; miR-21-5p, p = 0.002; miR-126-3p, p = 0.004 and miR-34a-5p, p < 0.005). Pathways regulated by these miRNAs include transforming growth factor beta (TGF-ß), Wnt, mammalian target of rapamycin (mTOR), Insulin, mitogen-activated protein kinase (MAPK) signaling, and senescence. Receiver operator characteristic (ROC) curve analysis indicated that miR-193a-5p (area under the curve AUC: 0.75) and miR-502-3p (AUC 0.71) have acceptable diagnostic value. This is the first identification of circulating miRNAs associated to ILA in respiratory asymptomatic subjects, providing potential non-invasive biomarkers and molecular targets to better understand the pathogenic mechanisms associated to ILA.

Downregulation of circulating miR-320a and target gene prediction in patients with diabetic retinopathy.

OBJECTIVE: To evaluate the expression of a set of miRNAs to identify differentially expressed miRNAs that might be considered reliable biomarkers on Diabetic Retinopathy (DR) blood samples. RESULTS: Expression levels of MiR-320a, MiR-342-3p, MiR-155, MiR-99a, MiR-29a and MiR-27b were analyzed in 60 healthy controls, 48 Diabetes Melitus (DM) without DR patients and 62 DR patients by qRT-PCR. MiR-320a was shown to be downregulated in the plasma of DR patients compared with DM patients without DR and healthy subjects. Target genes were predicted using miRWalk3.0, miR targeting data and target gene interaction data were imported to Cytoscape to visualize and merge networks and top ranked predicted genes were run through Ontology Genes to perform enrichment analysis on gene sets and classification system to identify biological processes and reactome pathways associated with DR. Highly scored target genes of miR-320a were categorized for various biological processes, including negative regulation of cell aging, negative regulation of cellular protein metabolic process and regulation of cellular response to stress that are critical to the development of DR. Our findings suggest that MiR-320a may have a role in the pathogenesis of DR and may represent novel biomarkers for this disease.

Identification of miRNAs Enriched in Extracellular Vesicles Derived from Serum Samples of Breast Cancer Patients.

MicroRNAs derived from extracellular vesicles (EV-miRNAs) are circulating miRNAs considered as potential new diagnostic markers for cancer that can be easily detected in liquid biopsies. In this study, we performed RNA sequencing analysis as a screening strategy to identify EV-miRNAs derived from serum of clinically well-annotated breast cancer (BC) patients from the south of Brazil. EVs from three groups of samples (healthy controls (CT), luminal A (LA), and triple-negative (TNBC)) were isolated from serum using a precipitation method and analyzed by RNA-seq (screening phase). Subsequently, four EV-miRNAs (miR-142-5p, miR-150-5p, miR-320a, and miR-4433b-5p) were selected to be quantified by quantitative real-time PCR (RT-qPCR) in individual samples (test phase). A panel composed of miR-142-5p, miR-320a, and miR-4433b-5p distinguished BC patients from CT with an area under the curve (AUC) of 0.8387 (93.33% sensitivity, 68.75% specificity). The combination of miR-142-5p and miR-320a distinguished LA patients from CT with an AUC of 0.9410 (100% sensitivity, 93.80% specificity). Interestingly, decreased expression of miR-142-5p and miR-150-5p were significantly associated with more advanced tumor grades (grade III), while the decreased expression of miR-142-5p and miR-320a was associated with a larger tumor size. These results provide insights into the potential application of EVs-miRNAs from serum as novel specific markers for early diagnosis of BC.

Liquid biopsy provides new insights into gastric cancer.

Liquid biopsies have great promise for precision medicine as they provide information about primary and metastatic tumors via a minimally invasive method. In gastric cancer patients, a large number of blood-based biomarkers have been reported for their potential role in clinical practice for screening, early diagnosis, prognostic evaluation, recurrence monitoring and therapeutic efficiency follow-up. This current review focuses on blood liquid biopsies' role and their clinical implications in gastric cancer patients, with an emphasis on circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) and circulating non-coding RNAs (ncRNAs). We also provide a brief discussion of the potential and limitations of liquid biopsies use and their future use in the routine clinical care of gastric cancer.

Translating microRNAs into biomarkers: What is new for pediatric cancer?

Since their discovery in 2008, cell-free circulating microRNAs have been considered potential biomarkers for various conditions, including pediatric cancer. Diagnosis of pediatric cancer still relies on clinical signs, which sometimes may be non-specific or appear at later stages. Thus, there is a need for a better understanding of molecules that allow a less invasive, early and effective method of cancer diagnosis. Despite the efforts of many researches to set specific miRNAs to be routinely used as diagnostic molecules, no miR has been currently utilized so far. In this study, we review the recent discoveries on circulating miRNAs in blood of patients suffering from the following pediatric cancers: osteosarcoma, rhabdomyosarcoma, Wilms tumor, acute myeloid leukemia, acute lymphocytic leukemia, retinoblastoma and neuroblastoma. We also focus on the roles of circulating miRs in tumorigenesis pathways, the methodological approaches used to detect and quantify circulating miRs, and discuss the challenges in using them routinely as biomarkers for pediatric cancers.

Circulating microRNA expression profiles in pre-eclampsia: evidence of increased miR-885-5p levels.

OBJECTIVE: To validate and to compare the circulating microRNA (miR) expression profiles between pre-eclampsia and healthy pregnant women, to perform correlation analysis of the differently expressed miRs with clinical and biochemical parameters, and to verify the extracellular localisation of miRs in apoptotic bodies, microvesicles, and exosomes. DESIGN: A case-control study with a replication study. SETTING: Pregnant women attending maternity hospitals in Southeastern Brazil. POPULATION: Two obstetric white populations: a case-control study (19 pre-eclampsia and 14 healthy pregnant) and a replication study (eight pre-eclampsia and eight healthy pregnant). METHODS: PCR-array with 84 different miRs was performed in plasma from five pre-eclampsia and four healthy pregnant women. In the case-control study, differently expressed miRs were validated using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and correlated with clinical and biochemical parameters. The plasma was then fractioned to study the extracellular localisation of miRs. MAIN OUTCOME MEASURES: Gene expression profiles of miRs. RESULTS: From PCR-array, three miRs (miR-376c-3p, miR-19a-3p, and miR-19b-3p) were found to be down-regulated and the miR-885-5p was found to be up-regulated in pre-eclampsia compared with healthy pregnant women. In the validation step, miR-885-5p was the only significantly different miR (fold-change = 5.0, P < 0.05), which was confirmed in the replication study (fold-change = 4.5, P < 0.05). Moreover, miR-885-5p was significantly correlated with the hepatic enzyme aspartate transaminase (r = 0.66; P = 0.0034) and it was mostly associated with the exosomes (32-fold higher than apoptotic bodies). CONCLUSIONS: miR-885-5p is increased in plasma from pre-eclampsia compared with healthy pregnant women, and it is released into circulation mainly inside exosomes. TWEETABLE ABSTRACT: miR-885-5p is increased in pre-eclampsia and is released into circulation mainly inside exosomes.

Circulating levels of miR-150 are associated with poorer outcomes of A/H1N1 infection.

BACKGROUND: Overproduction of pro-inflammatory cytokines and chemokines is frequently associated with severe clinical manifestations in patients infected with influenza A/H1N1 virus. Micro-RNAs (miRNAs) are highly conserved small non-coding RNA molecules that post-transcriptionally regulate gene expression and are potential biomarkers and therapeutic targets in different inflammatory conditions. METHODS: We studied the circulating and miRNA profiles in critically ill A/H1N1 patients, A/H1N1 patients with milder disease, asymptomatic housemates and healthy controls. Cytokine, chemokine and growth factors that were potential targets of differentially expressed miRNAs were assessed. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and interactome analysis of these miRNAs were also performed. RESULTS: Critically ill patients exhibited a significant over-expression of circulating miR-150 (p<0.005) when compared to patients with milder disease. miR-29c, miR-145 and miR-22 were differentially expressed in patients with severe A/H1N1 disease whereas miR-210, miR-126 and miR-222 were downregulated in individuals exposed to the A/H1N1 virus. Significant correlations (p<0.05) between circulating levels of miR-150 with IL-1ra, IL-2, IL-6, CXCL8, IFN-γ, CXCL10 and G-CSF were detected, particularly in critically ill patients. CONCLUSION: The up-regulation of miR-150 is associated with poorer outcomes of A/H1N1 infection. The differential expression of miRNAs related with immune processes in severe A/H1N1 disease supports the potential role of these miRNAs as biomarkers of disease progression.

Circulating profiling reveals the effect of a polyunsaturated fatty acid-enriched diet on common microRNAs.

BACKGROUND: Consumption of long-chain polyunsaturated fatty acids (PUFAs), which are abundant in seafood and nuts, ameliorates components of the metabolic syndrome. Circulating microRNAs (miRNAs) have demonstrated to be valuable biomarkers of metabolic diseases. Here, we investigated whether a sustained nuts-enriched diet can lead to changes in circulating miRNAs, in parallel to the dietary modification of fatty acids (FAs). METHODS AND RESULTS: The profile of 192 common miRNAs was assessed (TaqMan low-density arrays) in plasma from 10 healthy women before and after an 8-week trial with a normocaloric diet enriched with PUFAs (30 g/day of almonds and walnuts). The most relevant miRNAs were validated in an extended sample of 30 participants (8 men and 22 women). Adiponectin was measured by immunoassay and FAs by gas liquid chromatography coupled to mass spectrometry. The percentage of both ω-3 (P=.01) and ω-6 (P=.029) PUFAs of dietary origin (as inferred from plasma FA concentrations) increased, whereas saturated FAs decreased (P=.0008). Concomitantly with changes in circulating FAs, several miRNAs were modified by treatment, including decreased miR-328, miR-330-3p, miR-221 and miR-125a-5p, and increased miR-192, miR-486-5p, miR-19b, miR-106a, miR-769-5p, miR-130b and miR-18a. Interestingly, miR-106a variations in plasma correlated with changes in PUFAs, while miR-130b (r=0.58, P=.003) and miR-221 (r=0.46, P=.03) reflected changes in C-reactive protein. The dietary modulation of miR-125a-5p mirrored changes in fasting triglycerides (r=-0.44, P=.019) and increased adiponectin (r=0.43, P=.026). CONCLUSION: Dietary FAs (as inferred from plasma FA concentration) are linked to changes in circulating miRNAs, which may be modified by a PUFAs-enriched diet.

The diagnostic value of circulating microRNAs for middle-aged (40–60-year-old) coronary artery disease patients

OBJECTIVE: Circulating microRNAs have been recognized as promising biomarkers for various diseases. The present study aimed to explore the potential roles of circulating miR-149, miR-424 and miR-765 as non-invasive biomarkers for the diagnosis of coronary artery disease in middle-aged (40–60-year-old) patients. METHODS: Sixty-five stable coronary artery disease patients (49–57 years old), 30 unstable coronary artery disease patients (49–58 years old), and 32 non-coronary artery disease patients (49–-57 years old) who were matched for age, sex, smoking habits, hypertension and diabetes were enrolled in this study. Total RNA was isolated from plasma with TRIzol reagent. Circulating miRNA levels were measured by quantitative real-time polymerase chain reaction. RESULTS: Circulating miR-149 levels were decreased 4.49-fold in stable coronary artery disease patients (1.18 ± 0.84) and 5.09-fold in unstable coronary artery disease patients (1.04 ± 0.65) compared with non-coronary artery disease patients (5.30 ± 2.57) (p<0.001). Circulating miR-424 levels were reduced 3.6-fold in stable coronary artery disease patients (1.18 ± 0.60) and 5-fold in unstable coronary artery disease patients (0.86 ± 0.54) compared with non-coronary artery disease patients (4.35 ± 2.20) (p<0.001). In contrast, circulating miR-765 levels were elevated 3.98-fold in stable coronary artery disease patients (6.09 ± 2.27) and 5.33-fold in unstable coronary artery disease patients (8.17 ± 2.77) compared with non-coronary artery disease patients (1.53 ± 0.99) (p<0.001). Receiver operating characteristic curve analysis revealed that the respective areas under the curve for circulating miR-149, miR-424 and miR-765 were 0.938, 0.919 and 0.968 in stable CAD patients and 0.951, 0.960 and 0.977 in unstable coronary artery disease patients compared with non-coronary artery disease patients. CONCLUSION: Our results suggest that circulating miR-149, ...