Search

Activation of toll-like receptors (TLRs) plays a pivotal role in the host defense against bacteria and results in the activation of NF-κB-mediated transcription of proinflammatory mediators. Glucocorticoid-induced leucine zipper (GILZ) is an anti-inflammatory mediator, which inhibits NF-κB activity in macrophages. Thus, we aimed to investigate the regulation and role of GILZ expression in primary human and murine macrophages upon TLR activation. Treatment with TLR agonists, e.g., Pam3CSK4 (TLR1/2) or LPS (TLR4) rapidly decreased GILZ mRNA and protein levels. In consequence, GILZ downregulation led to enhanced induction of pro-inflammatory mediators, increased phagocytic activity, and a higher capacity to kill intracellular bacteria (Salmonella enterica serovar typhimurium), as shown in GILZ knockout macrophages. Treatment with the TLR3 ligand polyinosinic: polycytidylic acid [Poly(I:C)] did not affect GILZ mRNA levels, although GILZ protein expression was decreased. This effect was paralleled by sensitization toward TLR1/2- and TLR4-agonists. A bioinformatics approach implicated more than 250 miRNAs as potential GILZ regulators. Microarray analysis revealed that the expression of several potentially GILZ-targeting miRNAs was increased after Poly(I:C) treatment in primary human macrophages. We tested the ability of 11 of these miRNAs to target GILZ by luciferase reporter gene assays. Within this small set, four miRNAs (hsa-miR-34b*,−222,−320d,−484) were confirmed as GILZ regulators, suggesting that GILZ downregulation upon TLR3 activation is a consequence of the synergistic actions of multiple miRNAs. In summary, our data show that GILZ downregulation promotes macrophage activation. GILZ downregulation occurs both via MyD88-dependent and -independent mechanisms and can involve decreased mRNA or protein stability and an attenuated translation. [ABSTRACT FROM AUTHOR]

Since the benefit of prostate-specific antigen (PSA) screening remains controversial, new non-invasive biomarkers for prostate carcinoma (PCa) are still required. There is evidence that microRNAs (miRNAs) in whole peripheral blood can separate patients with localized prostate cancer from healthy individuals. However, the potential of blood-based miRNAs for the differential diagnosis of PCa and benign prostatic hyperplasia (BPH) has not been tested. We compared the miRNome from blood of PCa and BPH patients and further investigated the influence of the tumor volume, tumor-node-metastasis (TNM) classification, Gleason score, pretreatment risk status, and the pretreatment PSA value on the miRNA pattern. By microarray approach, we identified seven miRNAs that were significantly deregulated in PCa patients compared to BPH patients. Using quantitative real time PCR (qRT-PCR), we confirmed downregulation of hsa-miR-221* (now hsa-miR-221-5p) and hsa-miR-708* (now hsa-miR-708-3p) in PCa compared to BPH. Clinical parameters like PSA level, Gleason score, or TNM status seem to have only limited impact on the overall abundance of miRNAs in patients' blood, suggesting a no influence of these factors on the expression of deregulated miRNAs. [ABSTRACT FROM AUTHOR]

Background: The dependency of miRNA abundance from physiological processes such as exercises remains partially understood. We set out to analyze the effect of physical exercises on miRNA profiles in blood and plasma of endurance and strength athletes in a systematic manner and correlated differentially abundant miRNAs in athletes to disease miRNAs biomarkers towards a better understanding of how physical exercise may confound disease diagnosis by miRNAs.Methods: We profiled blood and plasma of 29 athletes before and after exercise. With four samples analyzed for each individual we analyzed 116 full miRNomes. The study set-up enabled paired analyses of individuals. Affected miRNAs were investigated for known disease associations using network analysis.Results: MiRNA patterns in blood and plasma of endurance and strength athletes vary significantly with differences in blood outreaching variations in plasma. We found only moderate differences between the miRNA levels before training and the RNA levels after training as compared to the more obvious variations found between strength athletes and endurance athletes. We observed significant variations in the abundance of miR-140-3p that is a known circulating disease markers (raw and adjusted p value of 5 × 10(-12) and 4 × 10(-7)). Similarly, the levels of miR-140-5p and miR-650, both of which have been reported as makers for a wide range of human pathologies significantly depend on the training mode. Among the most affected disease categories we found acute myocardial infarction. MiRNAs, which are up-regulated in endurance athletes inhibit VEGFA as shown by systems biology analysis of experimentally validated target genes.Conclusion: We provide evidence that the mode and the extent of training are important confounding factors for a miRNA based disease diagnosis. [ABSTRACT FROM AUTHOR]

Background: Circulating microRNAs (miRNAs) from blood are increasingly recognized as biomarker candidates for human diseases. Clinical routine settings frequently include blood sampling in tubes with EDTA as anticoagulant without considering the influence of phlebotomy on the overall miRNA expression pattern. We collected blood samples from six healthy individuals each in an EDTA blood collection tube. Subsequently, the blood was transferred into PAXgeneTM tubes at three different time points, i.e. directly (0 min), 10 min, and 2 h after phlebotomy. As control blood was also directly collected in PAXgeneTM blood RNA tubes that contain a reagent to directly lyse blood cells and stabilize their content. For all six blood donors at the four conditions (24 samples) we analyzed the abundance of 1,205 miRNAs by human Agilent miRNA V16 microarrays. Results: While we found generally a homogenous pattern of the miRNA abundance in all 24 samples, the duration of the EDTA treatment appears to influence the miRNA abundance of specific miRNAs. The most significant changes are observed after longer EDTA exposition. Overall, the impact of the different blood sample conditions on the miRNA pattern was substantially lower than intra-individual variations. While samples belonging to one of the six individuals mostly cluster together, there was no comparable clustering for any of the four tested blood sampling conditions. The most affected miRNA was miR-769-3p that was not detected in any of the six PAXgene blood samples, but in all EDTA 2h samples. Accordingly, hsa-miR-769-3p was also the only miRNA that showed a significantly different abundance between the 4 blood sample conditions by an ANOVA analysis (Benjamini-Hochberg adjusted p-value of 0.003). Validation by qRT-PCR confirmed this finding. Conclusion: The pattern of blood-borne miRNA abundance is rather homogenous between the four tested blood sample conditions of six blood donors. There was a clustering between the miRNA profiles that belong to a specific blood donor, but not between any of the four tested blood sampling conditions. The results show a limited overall impact of the blood sampling conditions on the miRNA pattern. Notwithstanding, the abundance of single miRNAs can be significantly altered by different blood sampling conditions. [ABSTRACT FROM AUTHOR]

Background: Neuromyelitis optica spectrum disorder (NMOSD) and multiple sclerosis (MS) have a similar clinical phenotype but represent distinct diseases, requiring different therapies. MicroRNAs (miRNAs) are short non-coding RNAs whose expression profiles can serve as diagnostic biomarkers and which may be involved in the pathophysiology of neuroinflammatory diseases. Here, we analyzed miRNA profiles in serum and whole blood of patients with NMOSD and clinically isolated syndrome (CIS)/relapsing-remitting MS (RRMS) as well as healthy controls by next-generation sequencing (NGS).Methods: MiRNA expression profiles were determined by NGS in sera of patients with aquaporin-4 antibody-positive NMOSD (n = 20), CIS/RRMS (n = 20), and healthy controls (n = 20) and in whole blood of patients with NMOSD (n = 11), CIS/RRMS (n = 60), and healthy controls (n = 43). Differentially expressed miRNAs were calculated by analysis of variance and t tests. All significance values were corrected for multiple testing. Selected miRNAs were validated in whole blood of patients with NMOSD (n = 18) and CIS/RRMS (n = 19) by quantitative real-time polymerase chain reaction (qRT-PCR).Results: None of 261 miRNAs detected in serum but 178 of 416 miRNAs detected in whole blood showed significantly different expression levels among the three groups. Pairwise comparisons revealed 115 (NMOSD vs. CIS/RRMS), 141 (NMOSD vs. healthy controls), and 44 (CIS/RRMS vs. healthy controls) miRNAs in whole blood with significantly different expression levels. qRT-PCR confirmed different expression levels in whole blood of patients with NMOSD and CIS/RRMS for 9 out of 10 exemplarily chosen miRNAs. In silico enrichment analysis demonstrated an accumulation of altered miRNAs in NMOSD in particular in CD15(+) cells (i.e., neutrophils and eosinophils).Conclusions: This study identifies a set of miRNAs in whole blood, which may have the potential to discriminate NMOSD from CIS/RRMS and healthy controls. In contrast, miRNA profiles in serum do not appear to be promising diagnostic biomarkers for NMOSD. Enrichment of altered miRNAs in CD15(+) neutrophils and eosinophils, which were previously implicated in the pathophysiology of NMOSD, suggests that miRNAs could be involved in the regulation of these cells in NMOSD. [ABSTRACT FROM AUTHOR]

Whole blood derived miRNA signatures determined by Next-Generation Sequencing (NGS) offer themselves as future minimally invasive biomarkers for various human diseases. The PAXgene system is a commonly used blood storage system for miRNA analysis. Central to all miRNA analyses that aim to identify disease specific miRNA signatures, is the question of stability and variability of the miRNA profiles that are generated by NGS. We characterized the influence of five different conditions on the genome wide miRNA expression pattern of human blood isolated in PAXgene RNA tubes. In detail, we analyzed 15 miRNomes from three individuals. The blood was subjected to different numbers of freeze/thaw cycles and analyzed for the influence of storage at -80 or 8 °C. We also determined the influence of blood collection and NGS preparations on the miRNA pattern isolated from a single individual, which has been sequenced 10 times. Here, five PAXGene tubes were consecutively collected that have been split in two replicates, representing two experimental batches. All samples were analyzed by Ulumina NGS. For each sample, approximately 20 million NGS reads have been generated. Hierarchical clustering and Principal Component Analysis (PCA) showed an influence of the different conditions on the miRNA patterns. The effects of the different conditions on miRNA abundance are, however, smaller than the differences that are due to interindividual variability. We alsofound evidence for an influence of the NGS measurement on the miRNA pattern. Specifically, hsa-mi-1271-5p and hsa-mi-182-5p showed coefficients of variation above 100% indicating a strong influence of the NGS protocol on the abundance of these miRNAs. [ABSTRACT FROM AUTHOR]

Background: While in the past decades nucleic acid analysis has been predominantly carried out using quantitative low- and high-throughput approaches such as qRT-PCR and microarray technology, next-generation sequencing (NGS) with its single base resolution is now frequently applied in DNA and RNA testing. Especially for small noncoding RNAs such as microRNAs there is a need for analysis and visualization tools that facilitate interpretation of the results also for clinicians. Methods: We developed miFRame, which supports the analysis of human small RNA NGS data. Our tool carries out different data analyses for known as well as predicted novel mature microRNAs from known precursors and presents the results in a well interpretable manner. Analyses include among others expression analysis of precursors and mature miRNAs, detection of novel precursors and detection of potential iso-microRNAs. Aggregation of results from different users moreover allows for evaluation whether remarkable results, such as novel mature miRNAs, are indeed specific for the respective experimental set-up or are frequently detected across a broad range of experiments. Results: We demonstrate the capabilities of miFRame, which is freely available at http://www.ccb.uni-saarland.de/ miframe on two studies, circulating biomarker screening for Multiple Sclerosis (cohort includes clinically isolated syndrome, relapse remitting MS, matched controls) as well as Alzheimer Disease (cohort includes Alzheimer Disease, Mild Cognitive Impairment, matched controls). Here, our tool allowed for an improved biomarker discovery by identifying likely false positive marker candidates. [ABSTRACT FROM AUTHOR]

Objectives: In our previous studies we reported a panel of 24 miRNAs that allowed discrimination between blood of lung tumor patients independent of the histological subtype and blood of healthy controls with an accuracy of 95.4% [94.9%-95.9%]. Here, we now separately analyzed the miRNA expression in blood of non-small cell lung cancer (NSCLC), including squamous cell lung cancer and adenocarcinoma, and small cell lung cancer (SCLC) patients. Patients and methods: In total, we examined the expression levels of 1,205 miRNAs in blood samples from 20 patients from each of the three histological groups and determined differentially expressed miRNAs between histological subtypes and metastatic and non-metastatic lung cancer. We further determined the overlap of miRNAs expressed in each subgroup with the 24-miRNA signature of lung tumor patients. Results: Based on a raw p-value < 0.05, only 18 blood-borne miRNAs were differentially expressed between patients with adenocarcinoma and with squamous cell lung carcinoma, 11 miRNAs between adenocarcinoma and SCLC, and 2 between squamous cell lung carcinoma and SCLC. Likewise, the comparison based on a fold change of 1.5 did not reveal major differences of the blood-borne miRNA expression pattern between NSCLC and SCLC. In addition, we found a large overlap between the blood-borne miRNAs detected in the three histological subgroups and the previously described 24-miRNA signature that separates lung cancer patients form controls. We identified several miRNAs that allowed differentiating between metastatic and non-metastatic tumors both in blood of patients with adenocarcinoma and in blood of patients with SCLC. Conclusion: There is a common miRNA expression pattern in blood of lung cancer patients that does not allow a reliable further subtyping into NSCLC or SCLC, or into adenocarcinoma and squamous cell lung cancer. The previously described 24-miRNA signature for lung cancer appears not primarily dependent on histological subtypes. However, metastatic adenocarcinoma and SCLC can be predicted with 75% accuracy. [ABSTRACT FROM AUTHOR]

Background MiRNAs from body fluids gain more and more attraction as biomarker candidates. Besides serum, patterns from whole blood are increasingly considered as markers for human pathologies. Usually, the contribution of different cell types to the respective signature remains however unknown. In this study we provide insights into the human miRNome of different compounds of the blood including CD3, CD14, CD15, CD19, CD56 positive cells as well as exosomes. Methods We measured the miRNA repertoire for each cell type and whole blood for two individuals at three time points over the course of one year in order to provide evidence that the cell type miRNomes can be reproducibly detected. Results For measurements repeated after 24 hours we found on average correlation of 0.97, even after one year profiles still correlated with 0.96, demonstrating the enormous stability of the cell type specific miRNomes. Highest correlation was found for CD15 positive cells, exceeding Pearson correlation of 0.99. For exosomes a significantly higher variability of miRNA expression was detected. In order to estimate the complexity and variability of the cell type specific miRNomes, we generated profiles for all considered cell types in a total of seven unaffected individuals. While CD15 positive cells showed the most complex miRNome consisting of 328 miRNAs, we detected significantly less miRNAs (186, p = 1.5*10-5) in CD19 positive cells. Moreover, our analysis showed functional enrichment in many relevant categories such as onco-miRNAs and tumor miRNA suppressors. Interestingly, exosomes were enriched just for onco-miRNAs but not for miRNA tumor suppressors. Conclusion In sum, our results provide evidence that blood cell type specific miRNomes are very consistent between individuals and over time. [ABSTRACT FROM AUTHOR]

Blood born micro(mi)RNA expression pattern have been reported for various human diseases with signatures specific for diseases. To evaluate these biomarkers, it is mandatory to know possible changes of miRNA signatures in healthy individuals under different physiological conditions. We analyzed the miRNA expression in peripheral blood of elite endurance athletes and moderatly active controls. Blood drawing was done before and after exhaustive exercise in each group. After Benjamini-Hochberg adjustment we did not find any miRNA with significant p-values when comparing miRNA expression between the different groups. We found, however, 24 different miRNAs with an expression fold change of minimum 1.5 in at least one of the comparisons (athletes before vs after exercise, athletes before exercise vs controls and athletes after exercise vs controls). The observed changes are not significant in contrast to the expression changes of the blood born miRNA expression reported for many human diseases. These data support the idea of disease associated miRNA patterns useful as biomarkers that are not readily altered by physiological conditions. [ABSTRACT FROM AUTHOR]

The article presents a comprehensive analysis of microRNA (miRNA) profiles in multiple sclerosis (MS), including next-generation sequencing (NGS). The analysis was performed in blood of patients with clinically isolated syndrome or relapsing-remitting multiple sclerosis. An overview of the methods of the study is given, along with a discussion on the results and conclusions of the comprehensive analysis.

Melanoma is the most aggressive form of skin cancer with estimated 48,000 deaths per year worldwide. The polyphenol curcumin derived from the plant Curcuma longa is well known for its anti-inflammatory and anti-cancerogenic properties. Accordingly, dietary intake of this compound may be suitable for melanoma prevention. However, how this compound affects basic cellular mechanisms in developing melanoma still remains elusive. Therefore, the aim of this study was to investigate for the first time the impact of oral curcumin administration on the miRNA signature of engrafting melanoma. For this purpose, the effects of a 4% curcumin diet were tested on melanoma, which were established by injection of murine B78H1 cells in the flank of C57BL/6 mice. Curcumin diet or standard chow (control) was administered two weeks prior to injection of tumor cells until termination of the experiment. High throughput chip-based array analysis was deployed to detect alterations in the miRNA signature of the tumors. Curcumin treatment significantly reduced the growth of the flank tumors. Furthermore the miRNA expression signature in tumors was substantially altered by curcumin intake with mmu-miR-205-5p over 100 times higher expressed when compared to controls. The expression levels of identified key miRNAs in the tumor samples were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). A comparable expression pattern of these miRNAs was also detected in other curcumin-treated melanoma cell lines under in vitro conditions. Putative targets of curcumin-induced up-regulated miRNAs were enriched in ‘o-glycan biosynthesis’, ‘endoplasmatic reticulum protein processing’ and different cancer-related pathways. Western Blot analyses revealed that of these targets anti-apoptotic B-cell CLL/lymphoma 2 (Bcl-2) and proliferating cell nuclear antigen (PCNA) were significantly down-regulated in curcumin-treated tumors. These findings demonstrate a profound alteration of the miRNA expression signature in engrafting curcumin-treated melanoma with mmu-miR-205-5p being up-regulated most significantly. [ABSTRACT FROM AUTHOR]

Little is known about the functions of miRNAs in human longevity. Here, we present the first genome-wide miRNA study in long-lived individuals (LLI) who are considered a model for healthy aging. Using a microarray with 863 miRNAs, we compared the expression profiles obtained from blood samples of 15 centenarians and nonagenarians (mean age 96.4 years) with those of 55 younger individuals (mean age 45.9 years). Eighty miRNAs showed aging-associated expression changes, with 16 miRNAs being up-regulated and 64 down-regulated in the LLI relative to the younger probands. Seven of the eight selected aging-related biomarkers were technically validated using quantitative RT-PCR, confirming the microarray data. Three of the eight miRNAs were further investigated in independent samples of 15 LLI and 17 younger participants (mean age 101.5 and 36.9 years, respectively). Our screening confirmed previously published miRNAs of human aging, thus reflecting the utility of the applied approach. The hierarchical clustering analysis of the miRNA microarray expression data revealed a distinct separation between the LLI and the younger controls ( P-value < 10−5). The down-regulated miRNAs appeared as a cluster and were more often reported in the context of diseases than the up-regulated miRNAs. Moreover, many of the differentially regulated miRNAs are known to exhibit contrasting expression patterns in major age-related diseases. Further in silico analyses showed enrichment of potential targets of the down-regulated miRNAs in p53 and other cancer pathways. Altogether, synchronized miRNA-p53 activities could be involved in the prevention of tumorigenesis and the maintenance of genomic integrity during aging. [ABSTRACT FROM AUTHOR]

Background: Blood-born miRNA signatures have recently been reported for various tumor diseases. Here, we compared the miRNA signature in Wilms tumor patients prior and after preoperative chemotherapy according to SIOP protocol 2001. Results: We did not find a significant difference between miRNA signature of both groups. However both, Wilms tumor patients prior and after chemotherapy showed a miRNA signature different from healthy controls. The signature of Wilms tumor patients prior to chemotherapy showed an accuracy of 97.5% and of patients after chemotherapy an accuracy of 97.0%, each as compared to healthy controls. Conclusion: Our results provide evidence for a blood-born Wilms tumor miRNA signature largely independent of four weeks preoperative chemotherapy treatment. [ABSTRACT FROM AUTHOR]

Abstract: Recently we reported differential miRNA signatures in blood cells of lung cancer patients and healthy controls. With the present study we wanted to investigate if miRNA blood signatures are also suited to differentiate lung cancer patients from COPD patients. We compared the expression of 863 human miRNAs in blood cells of lung cancer patients, COPD patients, and healthy controls. The miRNA pattern from patients with lung cancer and COPD were more similar to each other than to the healthy controls. However, we were able to discriminate lung cancer patients and COPD patients with 90.4% accuracy, 89.2% specificity, and 91.7% sensitivity. In total, 140 miRNAs were significant for the comparison COPD and controls, 61 miRNAs were significant for the comparison lung cancer and controls, and 14 miRNAs were significant for the comparison lung cancer and COPD. Screening target databases yielded over 400 putative targets for those 14 miRNAs. The predicted mRNA targets of three of the 14 miRNAs were significantly up-regulated in PBMCs of lung cancer patients compared to patients with non-malignant lung diseases. In conclusion, we showed that blood miRNA signatures are suitable to distinguish lung cancer from COPD. [Copyright &y& Elsevier]

Despite improved therapeutic regimens, primary CNS lymphoma (PCNSL) remains a therapeutic challenge. A prognostic classification of PCNSL patients may represent an important step towards optimised patient-adapted therapy. However, only higher age and low Karnofsky Performance Status (KPS) have repeatedly been reported to be associated with shorter overall survival (OS). Here we characterised microRNA (miRNA) fingerprints in the blood of PCNSL patients with short-term survival (STS) versus long-term survival (LTS) to assess their potential as novel prognostic biomarkers. Blood was collected from patients enrolled in the G-PCNSL-SG1 trial, a phase III study for patients with newly diagnosed PCNSL. miRNAs were extracted from the blood and analysed by next generation sequencing. The STS group comprised 20 patients with a median OS of 3 months and was compared to 20 LTS patients with a median OS of 55 months. The cohorts were balanced for age and KPS. Twelve annotated miRNAs were significantly deregulated between the two groups. Among them, miR-151a-5p and miR-151b exhibited the most prominent differences. Importantly, the combination of several miRNA allowed for a good separation between short- and long-term survivors with maximal Area Under Curve (AUC) above 0.75. Besides the known miRNAs we identified putative novel miRNA candidates with potential regulatory influence of PCNSL. Finally, the differential regulation of the most promising candidate miRNAs was confirmed by real-time polymerase chain reaction (PCR) in a validation cohort consisting of 20 STS and LTS patients. In conclusion, peripheral blood miRNA expression patterns hold promise as a prognostic tool in PCNSL patients. [ABSTRACT FROM AUTHOR]

Objective: To investigate the expression profiles of microRNA (miRNA) in human testes showing different histopathological patterns. Design: Microarray with quantitative real-time polymerase chain reaction (qRT-PCR) validation. Setting: University research and clinical institutes. Patient(s): Azoospermic men who underwent testicular biopsy for sperm recovery in preparation for intracytoplasmic sperm injection. Intervention(s): Testicular biopsies. Main Outcome Measure(s): Statistically significantly altered miRNA expression profiles among the testicular histopathologic patterns groups compared with normal pattern group. Result(s): According to miRNA array, a total of 197, 68, and 46 miRNAs were found to be differentially expressed when comparing the samples from Sertoli cell only (SCO), mixed atrophy (MA), and germ cell arrest (GA) groups, respectively, with normal spermatogenesis (N). Five miRNAs have been validated using qRT-PCR, and the results were consistent with miRNA array analysis. Bioinformatics analysis showed that five microRNAs (hsa-mir-34b*, hsa-mir-34b, hsa-mir-34c-5p, hsa-mir-449a, and hsa-mir-449b*) were involved in apoptosis, cell proliferation, and differentiation. Notably, potential target genes of these five miRNAs were involved in the spermatogenesis process. Conclusion(s): This study provides new insights into specific miRNAs that are expressed in infertile men with different histopathologic patterns, suggesting a role of miRNAs in regulating male germ and somatic cells and that their alteration is associated with reproductive abnormalities. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small noncoding RNA molecules, with essential functions in RNA silencing and post-transcriptional regulation of gene expression. miRNAs appear to regulate the development and function of the nervous system. Alterations of miRNA expression have been associated with Alzheimer's disease (AD). To characterize the AD miRNA signature, we examined genome-wide miRNA and mRNA expression patterns in the temporal cortex of AD and control samples. We validated our miRNA results by semiquantitative real-time polymerase chain reaction (PCR) in independent prefrontal cortex. Furthermore, we separated gray and white matter brain sections to identify the cellular origin of the altered miRNA expression. We observed genome-wide downregulation of hsa-miR-132-3p and hsa-miR-212-3p in AD with a stronger decrease in gray matter AD samples. We further identified 10 differently expressed transcripts achieving genome-wide levels of significance. Significantly deregulated miRNAs and mRNAs were correlated and examined for potential binding sites (in silico). This miRNome-wide study in AD provides supportive evidence and corroborates an important contribution of miR-132/212 and corresponding target mRNAs to the pathogenesis of AD. [ABSTRACT FROM AUTHOR]

Objective: To determine whether microRNA (miRNA) expression profile is different in extracellular microvesicles collected from seminal plasma of men with oligoasthenozoospermia, to gain further insight into molecular mechanisms underlying male infertility.Design: Microarray with quantitative real-time polymerase chain reaction validation and Western blot analysis confirmation.Setting: University research and clinical institutes.Patient(s): A total of 24 men, including 12 oligoasthenozoospermic subfertile men and 12 normozoospermic men.Intervention(s): None.Main Outcome Measure(s): Statistically significant altered miRNA expression profiles in oligoasthenozoospermic subfertile men compared with normozoospermic fertile men.Result(s): Extracellular microvesicles including exosomes were isolated from seminal plasma by ultracentrifugation. Presence of exosome-specific proteins was confirmed by Western blotting. In the extracellular microvesicles, we analyzed 1,205 miRNAs by microarray and identified 36 miRNAs with altered expression levels in oligoasthenozoospermic compared with normozoospermic fertile men. Seven miRNAs were overexpressed and 29 miRNAs were underexpressed in oligoasthenozoospermic men. Using quantitative real-time polymerase chain reaction as an independent method, we confirmed the significantly higher expression levels of miR-765 and miR-1275 and the significantly lower expression level of miR-15a in oligoasthenozoospermic subfertile men as compared with the normozoospermic men.Conclusion(s): We identified altered expression levels of miRNAs in extracellular microvesicles from seminal plasma as part of the molecular events in the male genital tract. These miRNAs may help to understand the molecular mechanisms underlying male infertility. [ABSTRACT FROM AUTHOR]

Objective To validate a set of five microRNAs (miRNAs) as specific biomarkers for the assessment of male infertility. Design Quantitative real-time polymerase chain reaction (qRT-PCR) validation study. Setting University research and clinical institutes. Patient(s) Two hundred twenty-six men presenting at an infertility clinic. Intervention(s) None. Main Outcome Measure(s) Validation analysis of a set of miRNAs in human purified spermatozoa and testicular biopsies. Result(s) Five miRNAs (hsa-miR-34b*, hsa-miR-34b, hsa-miR-34c-5p, hsa-miR-429, and hsa-miR-122) were confirmed with the use of qRT-PCR analysis in validation sets in patients with different forms of spermatogenic impairments (subfertile and nonobstructive azoospermia [NOA]) and control subjects. We found that hsa-miR-429 was significantly increased and the four other miRNAs were decreased in both tested groups compared with normal control subjects. Computing the area under the receiver operating characteristic curve (AUC) value for each of the five miRNAs, we showed that they separated the tested groups with high accuracy (range 0.777–0.988), except for hsa-miR-429 (AUC 0.475), in patient samples with NOA. Furthermore, with the use of support vector machine classification combining these five miRNAs, we found that they discriminated individuals with, respectively, subfertility and NOA from control subjects with an accuracy of 98.65% and 99.91%, a specificity of 98.44% and 99.69%, and a sensitivity of 98.83% and 100%. Conclusion(s) Our finding suggests that these five miRNAs have potential as novel noninvasive biomarkers to diagnose patients with subfertility. Except for hsa-miR-429, the combination of these miRNAs with other conventional tests would improve the diagnostic accuracy for detecting patients with different forms of NOA. [ABSTRACT FROM AUTHOR]

Objective: To determine whether microRNAs are differentially expressed in men with normal versus impaired spermatogenesis, and to find a biomarker for accurate diagnosis of male infertility. Design: Microarray with real-time polymerase chain reaction (RT-PCR) validation. Setting: University research and clinical institutes. Patient(s): Male partner of selected couples (n = 27) who were undergoing assisted reproduction techniques for infertility treatment. Intervention(s): None. Main Outcome Measure(s): Statistically significantly altered microRNA expression profiles in normozoospermic versus asthenozoospermic and oligoasthenozoospermic men. Result(s): There were 50 miRNAs up-regulated and 27 miRNAs down-regulated in asthenozoospermic males. In oligoasthenozoospermic males, 42 miRNAs were up-regulated and 44 miRNAs down-regulated when compared with normozoospermic males. The miRNAs that exhibited the highest fold changes and area under the receiver operating characteristic curve were miR-34b, miR-122, and miR-1973 in samples from asthenozoospermic men and miR-34b, miR-34b*, miR-15b, miR-34c-5p, miR-122, miR-449a, miR-1973, miR-16, and miR-19a in samples from oligoasthenozoospermic men. Furthermore, quantitative RT-PCR assays on specific miRNAs, including miR-141, miR-200a, miR-122, miR-34b, miR-34c-5p, and miR-16, yielded results that were largely consistent with the microarray data. Conclusion(s): Our results reveal an extended number of miRNAs that were differentially expressed in asthenozoospermic and oligoasthenozoospermic males compared with normozoospermic males. These data provide evidence for analysis of miRNA profiles as a future diagnosing tool for male infertility. [ABSTRACT FROM AUTHOR]