Bioinformatic identification and experimental validation of miRNAs from foxtail millet (Setariaitalica)

MiRNAs are a novel group of non-coding small RNAs that negatively regulate gene expression. Many miRNAs have been identified and investigated extensively in plant species with sequenced genomes. However, few miRNAs have been identified in foxtail millet (Setaria italica), which is an ancient cereal crop of great importance for dry land agriculture. In this study, 271 foxtail millet miRNAs belonging to 44 families were identified using a bioinformatics approach. Twenty-three pairs of sense/antisense miRNAs belonging to 13 families, and 18 miRNA clusters containing members of 8 families were discovered in foxtail millet. We identified 432 potential targets for 38 miRNA families, most of which were predicted to be involved in plant development, signal transduction, metabolic pathways, disease resistance, and environmental stress responses. Gene ontology (GO) analysis revealed that 101, 56, and 23 target genes were involved in molecular functions, biological processes, and cellular components, respectively. We investigated the expression patterns of 43 selected miRNAs using qRT-PCR analysis. All of the miRNAs were expressed ubiquitously with many exhibiting different expression levels in different tissues. We validated five predicted targets of four miRNAs using the RNA ligase mediated rapid amplification of cDNA end (5′-RLM-RACE) method.     

GPS: Identification of disease genes by rank aggregation of multi-genomic scoring schemes

In solving the gene prioritization problem, ranking candidate genes from most to least promising is attempted before further experimental validation. Integrating the results of various data sources and methods tends to result in a better performance when solving the gene prioritization problem. Therefore, a wide range of datasets and algorithms was investigated; these included topological features of protein networks, physicochemical characteristics and blast similarity scores of protein sequences, gene ontology, biological pathways, and tissue-based data sources. The novelty of this study lies in how the best-performing methods and reliable multi-genomic data sources were applied in an efficient two-step approach. In the first step, various multi-genomic data sources and algorithms were evaluated and seven best-performing rankers were then applied to prioritize candidate genes in different ways. In the second step, global prioritization was obtained by aggregating several scoring schemes.The results showed that protein networks, functional linkage networks, gene ontology, and biological pathway data sources have a significant impact on the quality of the gene prioritization approach. The findings also demonstrated a direct relationship between the degree of genes and the ranking quality of the evaluated tools. This approach outperformed previously published algorithms (e.g., DIR, GPEC, GeneDistiller, and Endeavour) in all evaluation metrices and led to the development of GPS software. Its user-friendly interface and accuracy makes GPS a powerful tool for the identification of human disease genes. GPS is available at http://gpsranker.com and http://LBB.ut.ac.ir.     

Identification of Wolbachia-responsive microRNAs in the two-spotted spider mite,Tetranychus urticae

Background

The two-spotted spider mite, Tetranychus urticae, is infected with Wolbachia, which have the ability to manipulate host reproduction and fitness. MicroRNAs (miRNAs) are small non-coding RNAs that are involved in many biological processes such as development, reproduction and host-pathogen interactions. Although miRNA was observed to involve in Wolbachia-host interactions in the other insect systems, its roles have not been fully deciphered in the two-spotted spider mite.

Results

Small RNA libraries of infected and uninfected T. urticae for both sexes (in total four libraries) were constructed. By integrating the mRNA data originated from the same samples, the target genes of the differentially expressed miRNAs were predicted. Then, GO and pathway analyses were performed for the target genes. Comparison of libraries showed that Wolbachia infection significantly regulated 91 miRNAs in females and 20 miRNAs in males, with an overall suppression of miRNAs in Wolbachia-infected libraries. A comparison of the miRNA and mRNA data predicted that the differentially expressed miRNAs negatively regulated 90 mRNAs in females and 9 mRNAs in males. An analysis of target genes showed that Wolbachia-responsive miRNAs regulated genes with function in sphingolipid metabolism, lysosome function, apoptosis and lipid transporting in both sexes, as well as reproduction in females.

Conclusion

Comparisons of the miRNA and mRNA data can help to identify miRNAs and miRNA target genes involving in Wolbachia-host interactions. The molecular targets identified in this study should be useful in further functional studies.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1122) contains supplementary material, which is available to authorized users.     

Insight into hypoxic preconditioning and ischemic injury through determination of nPKCε-interacting proteins in mouse brain

Cerebral hypoxic preconditioning (HPC) provides neuroprotection by intracellular signaling pathways. We previously demonstrated that novel protein kinase Cε (nPKCε) activation participated in cerebral HPC development. In this study, we explore the role of nPKCε in HPC-induced neuroprotection against middle cerebral artery occlusion (MCAO)-induced ischemic injury and identify its possible signaling molecules. A total of 131 adult male BALB/c mice were divided into eight groups: normoxic control (n = 9), HPC (n = 9), HPC + εV1–2 (n = 13), Sham (n = 19), HPC + sham (n = 6), Ischemia (I, 6 h MCAO, n = 31), HPC + I (n = 25) and HPC + εV1–2 + I (n = 19). nPKCε specific inhibitor εV1–2 was administered via intracerebroventricular injection. Western blot, 2,3,5-triphenyltetrazolium chloride staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling were applied to determine nPKCε membrane translocation, infarction volume and programmed cell death (PCD), respectively. Two-dimensional gel electrophoresis (2-De) and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to identify nPKCε-interacting proteins, followed by bioinformatics analysis of genee ontology (GO) to predict nPKCε-specific signaling pathways. Our results showed that HPC attenuates MCAO-induced brain injuries and stabilized nPKCεmembrane translocation in peri-infarct region, which was abolished by nPKCε-speecific inhibitor εV1–2. Proteomics analysis revealed 8 up- and 3 down-regulated nPKCε-interacting proteins both in cytosolic and particulate fractions of HPC mouse brain. GO analysis predicted 25 significant nPKCε-specific signaling pathways among the 16 identified nPKCε-interacting proteins in brain of HPC mice. This study is the first to report multiple nPKCε-interacting proteins and their signaling pathways in HPC mouse brain, suggesting that nPKCε signaling molecules is responsible for HPC-induced neuroprotection against cerebral ischemic injuries of mice.     

Analysis of microRNA expression profile induced by AICAR in mouse hepatocytes

AMP-activated protein kinase (AMPK) has been proposed to act as a key energy sensor mediating the metabolism of glucose and lipids, and pharmacological activation of AMPK may provide a new strategy for the management of type 2 diabetes. MicroRNAs (miRNAs) are a group of endogenous noncoding RNA that play important roles in many biological processes including energy metabolism. Whether miRNAs mediate AMPK action in regulating metabolic process is not clear. In this study, 0.5 mM 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) was added to increase activation of AMPK in 8 week old C57BL/6 mice primary hepatocytes. MiRNA microarray was performed to compare the miRNA expression profiles of hepatocytes treated with or without AICAR. We discovered that 41 miRNAs were significantly altered in AICAR-treated sample (fold change: > 2) compared with untreated control sample. Among them, 19 miRNAs were upregulated. MiRNA targets were predicted by TargetScan. Further bioinformatic analysis indicated that these predicted targets might be mainly involved in pathways of cellular metabolism and tumor pathogenesis. FUNDO analysis suggested that these predicted targets were enriched in cancer, diabetes mellitus, hypertension, obesity and heart failure (P < 0.01). A series of miRNAs could be regulated by the activation of AMPK and might mediate the action of AMPK during metabolic processes and tumor pathogenesis. Predicted target genes discovered in this study and pathway analysis provide new insights into hepatic metabolism and tumor pathogenesis regulated by AMPK signaling and clues to the possible molecular mechanism underlying the effect of AMPK.     

Inflammatory cytokines regulate microRNA-155 expression in human retinal pigment epithelial cells by activating JAK/STAT pathway

Inflammatory response of the retinal pigment epithelium plays a critical role in the pathogenesis of retinal degenerative diseases such as age-related macular degeneration. Our previous studies have shown that human retinal pigment epithelial (HRPE) cells, established from adult donor eyes, respond to inflammatory cytokines by enhancing the expression of a number of cytokines and chemokines. To investigate the role of microRNA (miRNA) in regulating this response, we performed microarray analysis of miRNA expression in HRPE cells exposed to inflammatory cytokine mix (IFN-γ + TNF-α + IL-1β). Microarray analysis revealed ∼11-fold increase in miR-155 expression, which was validated by real-time PCR analysis. The miR-155 expression was enhanced when the cells were treated individually with IFN-γ, TNF-α or IL-1β, but combinations of the cytokines exaggerated the effect. The increase in miR-155 expression by the inflammatory cytokines was associated with an increase in STAT1 activation as well as an increase in protein binding to putative STAT1 binding elements present in the MIR155 gene promoter region. All these activities were effectively blocked by JAK inhibitor 1. Our results show that the inflammatory cytokines increase miR-155 expression in human retinal pigment epithelial cells by activating the JAK/STAT signaling pathway.     

Deregulated microRNAs in CD4+ T cells from individuals with latent tuberculosis versus active tuberculosis

The mechanisms of latent tuberculosis (TB) infection remain elusive. Roles of microRNA (miRNA) have been highlighted in pathogen–host interactions recently. To identify miRNAs involved in the immune response to TB, expression profiles of miRNAs in CD4⁺ T cells from patients with latent TB, active TB and healthy controls were investigated by microarray assay and validated by RT‐qPCR. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used to analyse the significant functions and involvement in signalling pathways of the differentially expressed miRNAs. To identify potential target genes for miR‐29, interferon‐γ (IFN‐γ) mRNA expression was measured by RT‐qPCR. Our results showed that 27 miRNAs were deregulated among the three groups. RT‐qPCR results were generally consistent with the microarray data. We observed an inverse correlation between miR‐29 level and IFN‐γ mRNA expression in CD4⁺ T cells. GO and KEGG pathway analysis showed that the possible target genes of deregulated miRNAs were significantly enriched in mitogen‐activated protein kinase signalling pathway, focal adhesion and extracellular matrix receptor interaction, which might be involved in the transition from latent to active TB. In all, for the first time, our study revealed that some miRNAs in CD4⁺ T cells were altered in latent and active TB. Function and pathway analysis highlighted the possible involvement of miRNA‐deregulated mRNAs in TB. The study might help to improve understanding of the relationship between miRNAs in CD4⁺ T cells and TB, and laid an important foundation for further identification of the underlying mechanisms of latent TB infection and its reactivation.     

Pathway Network Analyses for Autism Reveal Multisystem Involvement,Major Overlaps with Other Diseases and Convergence upon MAPK and Calcium Signaling

We used established databases in standard ways to systematically characterize gene ontologies, pathways and functional linkages in the large set of genes now associated with autism spectrum disorders (ASDs). These conditions are particularly challenging—they lack clear pathognomonic biological markers, they involve great heterogeneity across multiple levels (genes, systemic biological and brain characteristics, and nuances of behavioral manifestations)—and yet everyone with this diagnosis meets the same defining behavioral criteria. Using the human gene list from Simons Foundation Autism Research Initiative (SFARI) we performed gene set enrichment analysis with the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Database, and then derived a pathway network from pathway-pathway functional interactions again in reference to KEGG. Through identifying the GO (Gene Ontology) groups in which SFARI genes were enriched, mapping the coherence between pathways and GO groups, and ranking the relative strengths of representation of pathway network components, we 1) identified 10 disease-associated and 30 function-associated pathways 2) revealed calcium signaling pathway and neuroactive ligand-receptor interaction as the most enriched, statistically significant pathways from the enrichment analysis, 3) showed calcium signaling pathways and MAPK signaling pathway to be interactive hubs with other pathways and also to be involved with pervasively present biological processes, 4) found convergent indications that the process “calcium-PRC (protein kinase C)-Ras-Raf-MAPK/ERK” is likely a major contributor to ASD pathophysiology, and 5) noted that perturbations associated with KEGG’s category of environmental information processing were common. These findings support the idea that ASD-associated genes may contribute not only to core features of ASD themselves but also to vulnerability to other chronic and systemic problems potentially including cancer, metabolic conditions and heart diseases. ASDs may thus arise, or emerge, from underlying vulnerabilities related to pleiotropic genes associated with pervasively important molecular mechanisms, vulnerability to environmental input and multiple systemic co-morbidities.     

High-Throughput Sequencing Identifies Novel and Conserved Cucumber (Cucumis sativus L.) microRNAs in Response to Cucumber Green Mottle Mosaic Virus Infection

Seedlings of Cucumis sativus L. (cv. ''Zhongnong 16'') were artificially inoculated with Cucumber green mottle mosaic virus (CGMMV) at the three-true-leaf stage. Leaf and flower samples were collected at different time points post-inoculation (10, 30 and 50 d), and processed by high throughput sequencing analysis to identify candidate miRNA sequences. Bioinformatic analysis using screening criteria, and secondary structure prediction, indicated that 8 novel and 23 known miRNAs (including 15 miRNAs described for the first time in vivo) were produced by cucumber plants in response to CGMMV infection. Moreover, gene expression profiles (p-value <0.01) validated the expression of 3 of the novel miRNAs and 3 of the putative candidate miRNAs and identified a further 82 conserved miRNAs in CGMMV-infected cucumbers. Gene ontology (GO) analysis revealed that the predicted target genes of these 88 miRNAs, which were screened using the psRNATarget and miRanda algorithms, were involved in three functional categories: 2265 in molecular function, 1362 as cellular components and 276 in biological process. The subsequent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the predicted target genes were frequently involved in metabolic processes (166 pathways) and genetic information processes (40 pathways) and to a lesser degree the biosynthesis of secondary metabolites (12 pathways). These results could provide useful clues to help elucidate host-pathogen interactions in CGMMV and cucumber, as well as for the screening of resistance genes.     

Enrichment Analysis Identifies Functional MicroRNA-Disease Associations in Humans

Substantial evidence has shown that microRNAs (miRNAs) may be causally linked to the occurrence and progression of human diseases. Herein, we conducted an enrichment analysis to identify potential functional miRNA-disease associations (MDAs) in humans by integrating currently known biological data: miRNA-target interactions (MTIs), protein-protein interactions, and gene-disease associations. Two contributing factors to functional miRNA-disease associations were quantitatively considered: the direct effects of miRNA that target disease-related genes, and indirect effects triggered by protein-protein interactions. Ninety-nine miRNAs were scanned for possible functional association with 2223 MeSH-defined human diseases. Each miRNA was experimentally validated to target ≥ 10 mRNA genes. Putative MDAs were identified when at least one MTI was confidently validated for a disease. Overall, 19648 putative MDAs were found, of which 10.0% was experimentally validated. Further results suggest that filtering for miRNAs that target a greater number of disease-related genes (n ≥ 8) can significantly enrich for true MDAs from the set of putative associations (enrichment rate = 60.7%, adjusted hypergeometric p = 2.41×10⁻⁹¹). Considering the indirect effects of miRNAs further elevated the enrichment rate to 72.6%. By using this method, a novel MDA between miR-24 and ovarian cancer was found. Compared with scramble miRNA overexpression of miR-24 was validated to remarkably induce ovarian cancer cells apoptosis. Our study provides novel insight into factors contributing to functional MDAs by integrating large quantities of previously generated biological data, and establishes a feasible method to identify plausible associations with high confidence.     

Dynamic expression of microRNAs in M2b polarized macrophages associated with systemic lupus erythematosus

Macrophage polarization contributes to the initiation and perpetuation of systemic lupus erythematosus (SLE). Our previous study demonstrated that M2b polarized macrophages induced by activated lymphocyte-derived DNA (ALD-DNA) have a crucial function in the initiation and progress of SLE disease. Accumulated data suggest that microRNAs (miRNAs) serve as critical regulators to control macrophage polarization. To investigate miRNA regulation during macrophage M2b polarization of SLE, miRNA microarrays of murine bone marrow derived macrophages (BMDMs) were performed following stimulation with ALD-DNA for 6 and 36 h. Over 11% of the 1111 analyzed miRNAs appeared differentially expressed during ALD-DNA triggered macrophage M2b polarization. Cluster analysis revealed certain patterns in miRNA expression that are closely linked to ALD-DNA induced macrophage M2b polarization. Analysis of the network structure showed that the predicted functions of the differentially regulated miRNAs at 6 h are significantly associated with inflammatory response and disease. Differentially regulated miRNAs identified at 36 h were determined to be significantly related to cell proliferation by biological network analysis. In this study, dynamic miRNA expression patterns and network analysis are described for the first time during ALD-DNA induced macrophage M2b polarization. The data not only provide a better understanding of miRNA-mediated macrophage polarization but also demonstrate the future therapeutic potential of targeting miRNAs in SLE patients.