Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages.

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.

MiR-148a impairs Ras/ERK signaling in B lymphocytes by targeting SOS proteins.

Although microRNAs have been recognized as central cellular regulators, there is an evident lack of knowledge about their targets. Here, we analyzed potential target genes for miR-148a functioning in Ras signaling in B cells, including SOS1 and SOS2. A dual-luciferase reporter assay showed significantly decreased luciferase activity upon ectopic overexpression of miR-148a in HEK-293T cells that were co-transfected with the 3'UTR of either SOS1 or SOS2. Each of the 3'UTRs of SOS1 and SOS2 contained two binding sites for miR-148a both of which were necessary for the decreased luciferase activity. MiR-148a overexpression in HEK-293T lead to significantly reduced levels of both endogenous SOS1 and SOS2 proteins. Likewise, reduced levels of SOS proteins were found in two B cell lines that were transfected with miR-148a. The level of ERK1/2 phosphorylation as one of the most relevant downstream members of the Ras/ERK signaling pathway was also reduced in cells with miR-148a overexpression. The data show that miR-148a impairs the Ras/ERK signaling pathway via SOS1 and SOS2 proteins in B cells.

Sources to variability in circulating human miRNA signatures.

An increasing number of studies propose circulating microRNAs (miRNAs) as biomarkers for a large number of human diseases including cancer, cardiovascular diseases, neurologic pathologies and others. To further validate miRNA as biomarkers it is indispensable to understand the variability of circulating miRNAs in healthy individuals. We determined the longitudinal miRNomes of 90 serum samples from the Janus Serum Bank in Norway, which have been stored between 23 and 40 y at -25 °Celsius. We profiled 3 serum samples with microarrays for 30 individuals, each. For each individual the samples were collected with a time interval of approximately 5 y. This design allowed insights into inter-individual variability, age dependent miRNA variability and the impact of storage length and pre-processing. A significant proportion of the miRNome was affected by the age of the blood donor and a not negligible, albeit small, part of the miRNome by the storage time. A substantial part of miRNAs was differentially abundant between individuals, independent of the time when samples were collected. Stepwise filtering of the 529 miRNAs that were detected in the serum samples showed 168 miRNAs with differential abundance depending on the time point analyzed, 56 miRNAs differentially abundant between individuals, and 169 miRNAs with an abundance depending on the sampling procedure. While these groups of miRNAs contain generally interesting and biologically important miRNAs, the remaining 135 miRNAs constitute very promising biomarker candidates as they show an overall low variability between healthy individuals, a likewise overall low variability across a longer life span, and a high independence of the sampling process and the storage length.

TRPC1- and TRPC3-dependent Ca2+ signaling in mouse cortical astrocytes affects injury-evoked astrogliosis in vivo.

Following brain injury astrocytes change into a reactive state, proliferate and grow into the site of lesion, a process called astrogliosis, initiated and regulated by changes in cytoplasmic Ca2+ . Transient receptor potential canonical (TRPC) channels may contribute to Ca2+ influx but their presence and possible function in astrocytes is not known. By RT-PCR and RNA sequencing we identified transcripts of Trpc1, Trpc2, Trpc3, and Trpc4 in FACS-sorted glutamate aspartate transporter (GLAST)-positive cultured mouse cortical astrocytes and subcloned full-length Trpc1 and Trpc3 cDNAs from these cells. Ca2+ entry in cortical astrocytes depended on TRPC3 and was increased in the absence of Trpc1. After co-expression of Trpc1 and Trpc3 in HEK-293 cells both proteins co-immunoprecipitate and form functional heteromeric channels, with TRPC1 reducing TRPC3 activity. In vitro, lack of Trpc3 reduced astrocyte proliferation and migration whereas the TRPC3 gain-of-function moonwalker mutation and Trpc1 deficiency increased astrocyte migration. In vivo, astrogliosis and cortex edema following stab wound injury were reduced in Trpc3-/- but increased in Trpc1-/- mice. In summary, our results show a decisive contribution of TRPC3 to astrocyte Ca2+ signaling, which is even augmented in the absence of Trpc1, in particular following brain injury. Targeted therapies to reduce TRPC3 channel activity in astrocytes might therefore be beneficial in traumatic brain injury.

EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research.

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

miRNAs in Ancient Tissue Specimens of the Tyrolean Iceman.

The analysis of nucleic acids in ancient samples is largely limited to DNA. Small noncoding RNAs (microRNAs) are known to be evolutionary conserved and stable. To gain knowledge on miRNAs measured from ancient samples, we profiled microRNAs in cryoconserved mummies. First, we established the approach on a World War One warrior, the "Kaiserjäger", which has been preserved for almost one century. Then, we profiled seven ancient tissue specimens including skeletal muscle, stomach mucosa, stomach content and two corpus organ tissues of the 5,300-year-old copper age mummy Iceman and compared these profiles to the presence of organ-specific miRNAs in modern tissues. Our analyses suggest the presence of specific miRNAs in the different Iceman's tissues. Of 1,066 analyzed human miRNAs, 31 were discovered across all biopsies and 87 miRNAs were detected only in a single sample. To check for potential microbiological contaminations, all miRNAs detected in Iceman samples and not present in ancient samples were mapped to 14,582 bacterial and viral genomes. We detected few hits (3.9% of miRNAs compared with 3.6% of miRNAs). Interestingly, the miRNAs with higher abundance across all ancient tissues were significantly enriched for Guanine (P value of 10-13) and Cytosine (P value of 10-7). The same pattern was observed for modern tissues. Comparing miRNAs measured from ancient organs to modern tissue patterns highlighted significant similarities, e.g., for miRNAs present in the muscle. Our first comprehensive analysis of microRNAs in ancient human tissues indicates that these stable molecules can be detected in tissue specimens after 5,300 years.

The miRNome of Alzheimer's disease: consistent downregulation of the miR-132/212 cluster.

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.

Paired proteomics, transcriptomics and miRNomics in non-small cell lung cancers: known and novel signaling cascades.

High-throughput omics analyses are applied to elucidate molecular pathogenic mechanisms in cancer. Given restricted cohort sizes and contrasting large feature sets paired multi-omics analysis supports discovery of true positive deregulated signaling cascades. For lung cancer patients we measured from the same tissue biopsies proteomic- (6,183 proteins), transcriptomic- (34,687 genes) and miRNomic data (2,549 miRNAs). To minimize inter-individual variations case and control lung biopsies have been gathered from the same individuals.Considering single omics entities, 15 of 2,549 miRNAs (0.6%), 752 of 34,687 genes (2.2%) and 141 of 6,183 proteins (2.3%) were significantly deregulated. Multivariate analysis also revealed that effects in miRNA were smaller compared to genes and proteins indicating that expression changes of miRNAs might also have limited impact of pathogenicity. However, a new algorithm for modeling the complex mutual interactions of miRNAs and their target genes facilitated precise prediction of deregulation in cancer genes (92.3% accuracy, p=0.007). Lastly, deregulation of genes in cancer matched deregulation of proteins coded by the genes in 80% of cases.The resulting interaction network, which is based on quantitative analysis of the abundance of miRNAs, mRNAs and proteins each taken from the same lung cancer tissue and from the same autologous normal lung tissue confirms molecular pathological changes and further contributes to the discovery of altered signaling cascades in lung cancer.

Altered micro-ribonucleic acid expression profiles of extracellular microvesicles in the seminal plasma of patients with oligoasthenozoospermia.

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.

miRNAs and sports: tracking training status and potentially confounding diagnoses.

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.

Identification of miR-34a-target interactions by a combined network based and experimental approach.

Circulating miRNAs have been associated with numerous human diseases. The lack of understanding the functional roles of blood-born miRNAs limits, however, largely their value as disease marker. In a systems biology analysis we identified miR-34a as strongly associated with pathogenesis. Genome-wide analysis of miRNAs in blood cell fractions highlighted miR-34a as most significantly up-regulated in CD3+ cells of lung cancer patients. By our in silico analysis members of the protein kinase C family (PKC) were indicated as miR-34a target genes. Using a luciferase assay, we confirmed binding of miR-34a-5p to target sequences within the 3'UTRs of five PKC family members. To verify the biological effect, we transfected HEK 293T and Jurkat cells with miR-34a-5p causing reduced endogenous protein levels of PKC isozymes. By combining bioinformatics approaches with experimental validation, we demonstrate that one of the most relevant disease associated miRNAs has the ability to control the expression of a gene family.

Syntaxin 8 is required for efficient lytic granule trafficking in cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) eliminate pathogen-infected and cancerous cells mainly by polarized secretion of lytic granules (LG, containing cytotoxic molecules like perforin and granzymes) at the immunological synapse (IS). Members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family are involved in trafficking (generation, transport and fusion) of vesicles at the IS. Syntaxin 8 (Stx8) is expressed in LG and colocalizes with the T cell receptor (TCR) upon IS formation. Here, we report the significance of Stx8 for human CTL cytotoxicity. We found that Stx8 mostly localized in late, recycling endosomal and lysosomal compartments with little expression in early endosomal compartments. Down-regulation of Stx8 by siRNA resulted in reduced cytotoxicity. We found that following perforin release of the pre-existing pool upon target cell contact, Stx8 down-regulated CTL regenerate perforin pools less efficiently and thus release less perforin compared to control CTL. CD107a degranulation, real-time and end-point population cytotoxicity assays, and high resolution microscopy support our conclusion that Stx8 is required for proper and timely sorting and trafficking of cytotoxic molecules to functional LG through the endosomal pathway in human CTL.

Integrated quantitative proteomic and transcriptomic analysis of lung tumor and control tissue: a lung cancer showcase.

Proteomics analysis of paired cancer and control tissue can be applied to investigate pathological processes in tumors. Advancements in data-independent acquisition mass spectrometry allow for highly reproducible quantitative analysis of complex proteomic patterns. Optimized sample preparation workflows enable integrative multi-omics studies from the same tissue specimens.We performed ion mobility enhanced, data-independent acquisition MS to characterize the proteome of 21 lung tumor tissues including adenocarcinoma and squamous cell carcinoma (SCC) as compared to control lung tissues of the same patient each. Transcriptomic data were generated for the same specimens. The quantitative proteomic patterns and mRNA abundances were subsequently analyzed using systems biology approaches.We report a significantly (p = 0.0001) larger repertoire of proteins in cancer tissues. 12 proteins were higher in all tumor tissues as compared to matching control tissues. Three proteins, CAV1, CAV2, and RAGE, were vice versa higher in all controls. We also identified characteristic SCC and adenocarcinoma protein patterns. Principal Component Analysis provided evidence that not only cancer from control tissue but also tissue from adenocarcinoma and SCC can be differentiated. Transcriptomic levels of key proteins measured from the same matched tissue samples correlated with the observed protein patterns.The applied study set-up with paired lung tissue specimens of which different omics are measured, is generally suited for an integrated multi-omics analysis.

Differential blood-based diagnosis between benign prostatic hyperplasia and prostate cancer: miRNA as source for biomarkers independent of PSA level, Gleason score, or TNM status.

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.

Deep characterization of blood cell miRNomes by NGS.

A systematic understanding of different factors influencing cell type specific microRNA profiles is essential for state-of-the art biomarker research. We carried out a comprehensive analysis of the biological variability and changes in cell type pattern over time for different cell types and different isolation approaches in technical replicates. All combinations of the parameters mentioned above have been measured, resulting in 108 miRNA profiles that were evaluated by next-generation-sequencing. The largest miRNA variability was due to inter-individual differences (34 %), followed by the cell types (23.4 %) and the isolation technique (17.2 %). The change over time in cell miRNA composition was moderate (<3 %) being close to the technical variations (<1 %). Largest variability (including technical and biological variance) was observed for CD8 cells while CD3 and CD4 cells showed significantly lower variations. ANOVA highlighted that 51.5 % of all miRNAs were significantly influenced by the purification technique. While CD4 cells were least affected, especially miRNA profiles of CD8 cells were fluctuating depending on the cell purification approach. To provide researchers access to the profiles and to allow further analyses of the tested conditions we implemented a dynamic web resource.

Distribution of miRNA expression across human tissues.

We present a human miRNA tissue atlas by determining the abundance of 1997 miRNAs in 61 tissue biopsies of different organs from two individuals collected post-mortem. One thousand three hundred sixty-four miRNAs were discovered in at least one tissue, 143 were present in each tissue. To define the distribution of miRNAs, we utilized a tissue specificity index (TSI). The majority of miRNAs (82.9%) fell in a middle TSI range i.e. were neither specific for single tissues (TSI > 0.85) nor housekeeping miRNAs (TSI < 0.5). Nonetheless, we observed many different miRNAs and miRNA families that were predominantly expressed in certain tissues. Clustering of miRNA abundances revealed that tissues like several areas of the brain clustered together. Considering -3p and -5p mature forms we observed miR-150 with different tissue specificity. Analysis of additional lung and prostate biopsies indicated that inter-organism variability was significantly lower than inter-organ variability. Tissue-specific differences between the miRNA patterns appeared not to be significantly altered by storage as shown for heart and lung tissue. MiRNAs TSI values of human tissues were significantly (P = 10(-8)) correlated with those of rats; miRNAs that were highly abundant in certain human tissues were likewise abundant in according rat tissues. We implemented a web-based repository enabling scientists to access and browse the data (https://ccb-web.cs.uni-saarland.de/tissueatlas).

Validating Alzheimer's disease micro RNAs using next-generation sequencing.

INTRODUCTION: Molecular biomarkers for Alzheimer's disease (AD) can support detection and improved care for patients, but novel candidates require verification. We previously reported a 12-micro RNA (miRNA) blood-based signature using next-generation sequencing (NGS) of 54 AD cases and 22 controls. METHODS: We performed validation of 49 AD cases and 55 controls using NGS and also included 20 mild cognitive impairment and 90 multiple sclerosis samples to identify nonspecific markers. Thus, 103 AD cases, 77 unaffected controls, and 110 diseased controls were sequenced. Although the initial cohort came predominantly from the United States, the validation samples were collected in Germany. RESULTS: Five hundred eighty miRNAs were detected in the blood. In the initial cohort, we observed 203, in the validation cohort, 146 dysregulated miRNAs at a significance level of 0.05. With 68 miRNAs, the overlap was significant (P = .0003). Likewise, the area under the receiver operator characteristic curve values of the miRNAs correlated (correlation of 0.93; 95% confidence interval 0.89-0.96; P <10(-16)). DISCUSSION: MiRNAs have the potential to support AD diagnosis and patient care.

DrugTargetInspector: An assistance tool for patient treatment stratification.

Cancer is a large class of diseases that are characterized by a common set of features, known as the Hallmarks of cancer. One of these hallmarks is the acquisition of genome instability and mutations. This, combined with high proliferation rates and failure of repair mechanisms, leads to clonal evolution as well as a high genotypic and phenotypic diversity within the tumor. As a consequence, treatment and therapy of malignant tumors is still a grand challenge. Moreover, under selective pressure, e.g., caused by chemotherapy, resistant subpopulations can emerge that then may lead to relapse. In order to minimize the risk of developing multidrug-resistant tumor cell populations, optimal (combination) therapies have to be determined on the basis of an in-depth characterization of the tumor's genetic and phenotypic makeup, a process that is an important aspect of stratified medicine and precision medicine. We present DrugTargetInspector (DTI), an interactive assistance tool for treatment stratification. DTI analyzes genomic, transcriptomic, and proteomic datasets and provides information on deregulated drug targets, enriched biological pathways, and deregulated subnetworks, as well as mutations and their potential effects on putative drug targets and genes of interest. To demonstrate DTI's broad scope of applicability, we present case studies on several cancer types and different types of input -omics data. DTI's integrative approach allows users to characterize the tumor under investigation based on various -omics datasets and to elucidate putative treatment options based on clinical decision guidelines, but also proposing additional points of intervention that might be neglected otherwise. DTI can be freely accessed at http://dti.bioinf.uni-sb.de.

Prioritizing and selecting likely novel miRNAs from NGS data.

Small non-coding RNAs play a key role in many physiological and pathological processes. Since 2004, miRNA sequences have been catalogued in miRBase, which is currently in its 21st version. We investigated sequence and structural features of miRNAs annotated in the miRBase and compared them between different versions of this reference database. We have identified that the two most recent releases (v20 and v21) are influenced by next-generation sequencing based miRNA predictions and show significant deviation from miRNAs discovered prior to the high-throughput profiling period. From the analysis of miRBase, we derived a set of key characteristics to predict new miRNAs and applied the implemented algorithm to evaluate novel blood-borne miRNA candidates. We carried out 705 individual whole miRNA sequencings of blood cells and collected a total of 9.7 billion reads. Using miRDeep2 we initially predicted 1452 potentially novel miRNAs. After excluding false positives, 518 candidates remained. These novel candidates were ranked according to their distance to the features in the early miRBase versions allowing for an easier selection of a subset of putative miRNAs for validation. Selected candidates were successfully validated by qRT-PCR and northern blotting. In addition, we implemented a web-server for ranking potential miRNA candidates, which is available at:www.ccb.uni-saarland.de/novomirank.