Understanding Of miRNA Function Research Articles

Experimental design and data analysis of Ago-RIP-Seq experiments for the identification of microRNA targets.

The identification of microRNA (miRNA) target genes is crucial for understanding miRNA function. Many methods for the genome-wide miRNA target identification have been developed in recent years; however, they have several limitations including the dependence on low-confident prediction programs and artificial miRNA manipulations. Ago-RNA immunoprecipitation combined with high-throughput sequencing (Ago-RIP-Seq) is a promising alternative. However, appropriate statistical data analysis algorithms taking into account the experimental design and the inherent noise of such experiments are largely lacking.Here, we investigate the experimental design for Ago-RIP-Seq and examine biostatistical methods to identify de novo miRNA target genes. Statistical approaches considered are either based on a negative binomial model fit to the read count data or applied to transformed data using a normal distribution-based generalized linear model. We compare them by a real data simulation study using plasmode data sets and evaluate the suitability of the approaches to detect true miRNA targets by sensitivity and false discovery rates. Our results suggest that simple approaches like linear regression models on (appropriately) transformed read count data are preferable.

Large-scale analysis of microRNA expression, epi-transcriptomic features and biogenesis.

MicroRNAs are important genetic regulators in both animals and plants. They have a range of functions spanning development, differentiation, growth, metabolism and disease. The advent of next-generation sequencing technologies has made it a relatively straightforward task to detect these molecules and their relative expression via sequencing. There are a large number of published studies with deposited datasets. However, there are currently few resources that capitalize on these data to better understand the features, distribution and biogenesis of miRNAs. Herein, we focus on Human and Mouse for which the majority of data are available. We reanalyse sequencing data from 461 samples into a coordinated catalog of microRNA expression. We use this to perform large-scale analyses of miRNA function and biogenesis. These analyses include global expression comparison, co-expression of miRNA clusters and the prediction of miRNA strand-specificity and underlying constraints. Additionally, we report for the first time a global analysis of miRNA epi-transcriptomic modifications and assess their prevalence across tissues, samples and families. Finally, we report a list of potentially mis-annotated miRNAs in miRBase based on their aggregated modification profiles. The results have been collated into a comprehensive online repository of miRNA expression and features such as modifications and RNA editing events, which is available at: http://wwwdev.ebi.ac.uk/enright-dev/miratlas. We believe these findings will further contribute to our understanding of miRNA function in animals and benefit the miRNA community in general.

A network-biology perspective of microRNA function and dysfunction in cancer.

MicroRNAs (miRNAs) participate in most aspects of cellular differentiation and homeostasis, and consequently have roles in many pathologies, including cancer. These small non-coding RNAs exert their effects in the context of complex regulatory networks, often made all the more extensive by the inclusion of transcription factors as their direct targets. In recent years, the increased availability of gene expression data and the development of methodologies that profile miRNA targets en masse have fuelled our understanding of miRNA functions, and of the sources and consequences of miRNA dysregulation. Advances in experimental and computational approaches are revealing not just cancer pathways controlled by single miRNAs but also intermeshed regulatory networks controlled by multiple miRNAs, which often engage in reciprocal feedback interactions with the targets that they regulate.

MicroRNA Transcriptome of Poly I:C-Stimulated Peripheral Blood Mononuclear Cells Reveals Evidence for MicroRNAs in Regulating Host Response to RNA Viruses in Pigs.

MicroRNAs (miRNAs) are one family of small noncoding RNAs that function to modulate the activity of specific mRNA targets in animals. To understand the role of miRNAs in regulating genes involved in the host immune response to RNA viruses, we profiled and characterized the miRNAs of swine peripheral blood mononuclear cells (PBMC) stimulated with poly I:C, a synthetic dsRNA analog, by miRNA-sequencing (miRNA-seq). We identified a total of 905 miRNAs, of which 503 miRNAs were firstly exploited herein with no annotation in the latest miRBase 21.0. Expression analysis demonstrated that poly I:C stimulation can elicit significantly differentially expressed (DE) miRNAs in Dapulian (n = 20), one Chinese indigenous breed, as well as Landrace (n = 23). By integrating the mRNA expression profiles of the same sample with miRNA profiles, we carried out function analyses of the target genes of these DE miRNAs, with the results indicating that target genes were most enriched in some immune-related pathways and gene ontology (GO) terms, suggesting that DE miRNAs play an important role in the regulation of host to poly I:C stimulation. Furthermore, we also detected 43 and 61 significantly DE miRNAs between the two breeds in the control sample groups and poly I:C stimulation groups, respectively, which may be involved in regulation of the different characteristics of the two breeds. This study describes for the first time the PBMC miRNA transcriptomic response to poly I:C stimulation in pigs, which not only contributes to a broad view of the pig miRNAome but improves our understanding of miRNA function in regulating host immune response to RNA viruses.

Visualization and Quantification of MicroRNA in a Single Cell Using Atomic Force Microscopy.

MicroRNAs (miRNAs) play critical roles in controlling various cellular processes, and the expression levels of individual miRNAs can be considerably altered in pathological conditions such as cancer. Accurate quantification of miRNA at the single-cell level will lead to a better understanding of miRNA function. Here, we present a direct and sensitive method for miRNA detection using atomic force microscopy (AFM). A hybrid binding domain (HBD)-tethered tip enabled mature miRNAs, but not premature miRNAs, to be located individually on an adhesion force map. By scanning several sections of a micrometer-sized DNA spot, we were able to quantify the copy number of miR-134 in a single neuron and demonstrate that the expression was increased upon cell activation. Moreover, we visualized individual miR-134s on fixed neurons after membrane removal and observed 2-4 miR-134s in the area of 1.0 × 1.0 μm(2) of soma. The number increased to 8-14 in stimulated neurons, and this change matches the ensemble-averaged increase in copy number. These findings indicate that miRNAs can be reliably quantified at the single cell level with AFM and that their distribution can be mapped at nanometric lateral resolution without modification or amplification. Furthermore, the analysis of miRNAs, mRNAs, and proteins in the same sample or region by scanning sequentially with different AFM tips would let us accurately understand the post-transcriptional regulation of biological processes.

MiRSEA: Discovering the pathways regulated by dysfunctional MicroRNAs.

Recent studies have shown that dysfunctional microRNAs (miRNAs) are involved in the progression of various cancers. Dysfunctional miRNAs may jointly regulate their target genes and further alter the activities of canonical biological pathways. Identification of the pathways regulated by a group of dysfunctional miRNAs could help uncover the pathogenic mechanisms of cancer and facilitate development of new drug targets. Current miRNA-pathway analyses mainly use differentially-expressed miRNAs to predict the shared pathways on which they act. However, these methods fail to consider the level of differential expression level, which could improve our understanding of miRNA function. We propose a novel computational method, MicroRNA Set Enrichment Analysis (MiRSEA), to identify the pathways regulated by dysfunctional miRNAs. MiRSEA integrates the differential expression levels of miRNAs with the strength of miRNA pathway associations to perform direct enrichment analysis using miRNA expression data. We describe the MiRSEA methodology and illustrate its effectiveness through analysis of data from hepatocellular cancer, gastric cancer and lung cancer. With these analyses, we show that MiRSEA can successfully detect latent biological pathways regulated by dysfunctional miRNAs. We have implemented MiRSEA as a freely available R-based package on CRAN (https://cran.r-project.org/web/packages/MiRSEA/).

MiRNA Profile in Three Different Normal Human Ocular Tissues by miRNA-Seq.

PurposeBecause microRNAs (miRNAs) have been associated with eye diseases, our study aims to profile ocular miRNA expression in normal human ciliary body (CB), cornea, and trabecular meshwork (TM) using miRNA-Seq to provide a foundation for better understanding of miRNA function and disease involvement in these tissues.MethodsTotal RNAs were extracted from seven normal human CB, seven cornea, and seven TM samples using mirVana total RNA isolation kit. miRNA-Seq was done with Illumina MiSeq. Bowtie software was used to trim and align generated sequence reads, and only exact matches to mature miRNAs from miRBase were included. The miRTarBase database was used to analyze miRNA target interactions, and the expression of five selected miRNAs was validated using droplet digital PCR (ddPCR).ResultsUsing the miRNA extracted from 21 human samples, we found 378 miRNAs collectively expressed, of which the 11 most abundant miRNAs represented 80% of the total normalized reads. We also identified uniquely expressed miRNAs, of which five share 18 highly validated gene targets, and created a profile of miRNAs known to target genes associated with keratoconus and glaucoma. Using ddPCR, we validated the expression profile of five miRNAs from miRNA-Seq.ConclusionsFor the first time, we profiled miRNA expression in three human ocular tissues using miRNA-Seq, identifying many miRNAs that had not been previously reported in ocular tissue. Defining the relative expression of miRNAs in nondiseased eye tissues could help uncover changes in miRNA expression that accompany diseases such as glaucoma and keratoconus.

Genome‐wide identification and characterization of Eutrema salsugineummicroRNAs for salt tolerance

Eutrema salsugineum, a close relative of Arabidopsis thaliana, is a valuable halophytic model plant that has extreme tolerance to salinity. As posttranscriptional gene regulators, microRNAs (miRNAs) control gene expression and a variety of biological processes, including plant-stress responses. To identify salt-stress responsive miRNAs in E. salsugineum and reveal their possible roles in the adaptive response to salt stress, we chose the Solexa sequencing platform to screen the miRNAs in 4-week-old E. salsugineum seedlings under salt treatment. A total of 82 conserved miRNAs belonging to 27 miRNA families and 17 novel miRNAs were identified and 11 conserved miRNA families and 4 novel miRNAs showed a significant response to salt stress. To investigate the possible biological roles of miRNAs, 1060 potential targets were predicted. Moreover, 35 gene ontology (GO) categories and 1 pathway, including a few terms that were directly and indirectly related to salt stress, were significantly enriched in the salt-stress-responsive miRNAs targets. The relative expression analysis of six target genes was analyzed using quantitative real-time polymerase chain reaction (PCR) and showed a negative correlation with their corresponding miRNAs. Many stress regulatory and phytohormone regulatory cis-regulatory elements were widely present in the promoter region of the salt-responsive miRNA precursors. This study describes the large-scale characterization of E. salsugineum miRNAs and provides a useful resource for further understanding of miRNA functions in the regulation of the E. salsugineum salt-stress response.

Capture and Identification of miRNA Targets by Biotin Pulldown and RNA-seq.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate the stability and expression of target RNAs in a sequence-dependent manner. Identifying miRNA-regulated genes is key to understanding miRNA function. Here, we describe an unbiased biochemical pulldown method to identify with high-specificity miRNA targets. Regulated transcripts are enriched in streptavidin-captured mRNAs that bind to a transfected biotinylated miRNA mimic. The method is relatively simple, does not involve cross-linking and can be performed with only a million cells. Addition of an on-bead RNase digestion step also identifies miRNA recognition elements (MRE).

MicroRNA-15b/16 Enhances the Induction of Regulatory T Cells by Regulating the Expression of Rictor and mTOR.

CD4(+) regulatory T cells (Tregs) are essential for controlling immune responses and preventing autoimmunity. Their development requires regulation of gene expression by microRNAs (miRNAs). To understand miRNA function in Treg development, we searched for important miRNAs and their relevant target genes. Of the more abundantly expressed miRNAs in Tregs, only miR-15b/16, miR-24, and miR-29a impacted the production of in vitro-induced Tregs (iTregs) in overexpression and blocking experiments. miRNA mimics for these significantly enhanced the induction of iTregs in Dicer(-/-) CD4(+) T cells. Furthermore, the overexpression of miR-15b/16 in conventional CD4(+) T cells adoptively transferred into Rag2(-/-) mice increased the in vivo development of peripheral Tregs and diminished the severity of autoimmune colitis. In searching for targets of miR-15b/16, we observed that the mammalian target of rapamycin (mTOR) signaling pathway was enhanced in Dicer(-/-) CD4(+) T cells, and its pharmacological inhibition restored induction of iTregs. Suppression of mTOR signaling is essential for induction of iTregs from naive CD4(+) T cells, and the mTORC2 component, Rictor, contained a functional target site for miR-15b/16. Rictor was more abundantly expressed in Dicer(-/-) T cells as was mTOR, and their expression was downregulated by the overexpression of miR-15b/16. This led to a reduction in mTOR signaling, as measured by phosphorylation of the downstream target, ribosomal protein S6. Finally, knockdown of Rictor by small interfering RNAs enhanced Treg induction in Dicer(-/-) CD4(+) T cells. Therefore, an important mechanism of miRNA regulation of Treg development is through regulation of the mTOR signaling pathway.

Spatiotemporal analysis of microRNA-8 reveals important role in mosquito reproductive processes

Female mosquitoes require a blood meal for reproduction, providing the underlying mechanism for the spread of many devastating vector-borne diseases in humans. Understanding the mechanisms that govern the major functions linked to the female mosquito's ability to utilize blood and develop eggs is of paramount importance. Reports have indicated that microRNAs (miRNAs) are differentially expressed in various tissues of the female mosquito upon the uptake of a blood meal. In our previous research, we have reported the importance of miRNAs in regulating mosquito blood digestion through the characterization of the conserved miRNA, miR-275, and the mosquito specific miRNA, miR-1174. Our most recent work has shown that the conserved miRNA, miR-8, targets the Wingless signaling pathway to regulate secretion of yolk protein precursors by the female mosquito fat body and accumulation into the developing ovaries. Here, we summarize the role of miRNAs in the female mosquito, particularly miR-8. We also discuss the recent advances in mosquito biology and how these genetic tools can enhance our understanding of miRNA function.

Analysis of MicroRNA Expression Profiles in Weaned Pig Skeletal Muscle after Lipopolysaccharide Challenge.

MicroRNAs (miRNAs) constitute a class of non-coding RNAs that play a crucial regulatory role in skeletal muscle development and disease. Several acute inflammation conditions including sepsis and cancer are characterized by a loss of skeletal muscle due primarily to excessive muscle catabolism. As a well-known inducer of acute inflammation, a lipopolysaccharide (LPS) challenge can cause serious skeletal muscle wasting. However, knowledge of the role of miRNAs in the course of inflammatory muscle catabolism is still very limited. In this study, RNA extracted from the skeletal muscle of pigs injected with LPS or saline was subjected to small RNA deep sequencing. We identified 304 conserved and 114 novel candidate miRNAs in the pig. Of these, four were significantly increased in the LPS-challenged samples and five were decreased. The expression of five miRNAs (ssc-miR-146a-5p, ssc-miR-221-5p, ssc-miR-148b-3p, ssc-miR-215 and ssc-miR-192) were selected for validation by quantitative polymerase chain reaction (qPCR), which found that ssc-miR-146a-5p and ssc-miR-221-5p were significantly upregulated in LPS-challenged pig skeletal muscle. Moreover, we treated mouse C2C12 myotubes with 1000 ng/mL LPS as an acute inflammation cell model. Expression of TNF-α, IL-6, muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1) mRNA was strongly induced by LPS. Importantly, miR-146a-5p and miR-221-5p also showed markedly increased expression in LPS-treated C2C12 myotubes, suggesting the two miRNAs may be involved in muscle catabolism systems in response to acute inflammation caused by a LPS challenge. To our knowledge, this study is the first to examine miRNA expression profiles in weaned pig skeletal muscle challenged with LPS, and furthers our understanding of miRNA function in the regulation of inflammatory muscle catabolism.

Identification of MicroRNA for Intermuscular Bone Development in Blunt Snout Bream (Megalobrama amblycephala).

Intermuscular bone (IB), which occurs only in the myosepta of the lower teleosts, is attracting more attention of researchers due to its particular development and lack of genetic information. MicroRNAs (miRNAs) are emerging as important regulators for biological processes. In the present study, miRNAs from IBs and connective tissue (CT; encircled IBs) from six-month-old Megalobrama amblycephala were characterized and compared. The results revealed the sequences and expression levels of 218 known miRNA genes (belonging to 97 families). Of these miRNAs, 44 known microRNA sequences exhibited significant expression differences between the two libraries, with 24 and 20 differentially-expressed miRNAs exhibiting higher expression in the CT and IBs libraries, respectively. The expressions of 11 miRNAs were selected to validate in nine tissues. Among the high-ranked predicted gene targets, differentiation, cell cycle, metabolism, signal transduction and transcriptional regulation were implicated. The pathway analysis of differentially-expressed miRNAs indicated that they were abundantly involved in regulating the development and differentiation of IBs and CT. This study characterized the miRNA for IBs of teleosts for the first time, which provides an opportunity for further understanding of miRNA function in the regulation of IB development.

Micro RNA research in cattle, pig, sheep, and chicken

MicroRNAs (miRNAs) are small non-coding RNAs that play key roles in regulating gene expression. Polymorphisms in miRNA precursors, target genes or within components of silencing machinery contribute significantly to the phenotypic diversity in animals. Due to this role miRNAs became the subject of increased research interest in association with production traits in livestock. In this article we presented examples of associations between miRNA genes and phenotypes of four livestock species: cattle, pig, sheep, and chicken. Most miRNA research studies are focused on their functioning in muscle, adipose tissues, gonads, fetal development and immune system. MicroRNA functions also impact animal productivity and consequently economic success of farming. With understanding miRNA functions in various biological pathways it is possible to develop new strategies for improving the productivity of livestock.

Seeing through the miRage of tissue complexity

Seeing through the miRage of tissue complexity

Sequencing of Captive Target Transcripts Identifies the Network of Regulated Genes and Functions of Primate-Specific miR-522

Identifying microRNA (miRNA)-regulated genes is key to understanding miRNA function. However, many miRNA recognition elements (MREs) do not follow canonical "seed" base-pairing rules, making identification of bona fide targets challenging. Here, we apply an unbiased sequencing-based systems approach to characterize miR-522, a member of the oncogenic primate-specific chromosome 19 miRNA cluster, highly expressed in poorly differentiated cancers. To identify miRNA targets, we sequenced full-length transcripts captured by a biotinylated miRNA mimic. Within these targets, mostly noncanonical MREs were identified by sequencing RNase-resistant fragments. miR-522 overexpression reduced mRNA, protein levels, and luciferase activity of >70% of a random list of candidate target genes and MREs. Bioinformatic analysis suggested that miR-522 regulates cell proliferation, detachment, migration, and epithelial-mesenchymal transition. miR-522 induces G1 cell-cycle arrest and causes cells to detach without anoikis, become invasive, and express mesenchymal genes. Thus, our method provides a simple but effective technique for identifying miRNA-regulated genes and biological function.

MicroRNAs in human lung cancer.

Lung cancer, which can be divided into two major clinical-pathological categories, small cell lung cancer and non-small cell lung cancer, is the leading cause of cancer-related death worldwide. MicroRNAs (miRNAs), small non-coding RNAs approximately 22 nucleotides in length, have been reported to be upregulated or downregulated in disease states and specific cell types. Recently, miRNAs have gained recognition as major regulators of human gene expression. MiRNAs can control highly complex signal transduction pathways and other biological pathways by targeting and controlling gene expression, accounting for their important role in lung cancer. Findings from recent studies on the roles of miRNAs in lung cancer are summarized in this review. Understanding miRNA functions in lung cancer will bring molecular-level insight leading to better prognosis, diagnosis, and therapeutic approaches.

Transcriptome-wide Discovery of microRNA Binding Sites in Human Brain

The orchestration of brain function requires complex gene regulatory networks that are modulated, in part, by microRNAs (miRNAs). These noncoding RNAs associate with argonaute (Ago) proteins in order to direct posttranscriptional gene suppression via base pairing with target transcripts. In order to better understand how miRNAs contribute to human-specialized brain processes and neurological phenotypes, identifying their targets is of paramount importance. Here, we address the latter by profiling Ago2:RNA interactions using HITS-CLIP to generate a transcriptome-wide map of miRNA binding sites in human brain. We uncovered ∼ 7,000 stringent Ago2 binding sites that are highly enriched for conserved sequences corresponding to abundant brain miRNAs. This interactome points to functional miRNA:target pairs across >3,000 genes and represents a valuable resource for accelerating our understanding of miRNA functions in brain. We demonstrate the utility of this map for exploring clinically relevant miRNA binding sites that may facilitate the translation of genetic studies of complex neuropsychiatric diseases into therapeutics.

Large-scale investigation of human TF-miRNA relations based on coexpression profiles.

Noncoding, endogenous microRNAs (miRNAs) are fairly well known for regulating gene expression rather than protein coding. Dysregulation of miRNA gene, either upregulated or downregulated, may lead to severe diseases or oncogenesis, especially when the miRNA disorder involves significant bioreactions or pathways. Thus, how miRNA genes are transcriptionally regulated has been highlighted as well as target recognition in recent years. In this study, a large-scale investigation of novel cis- and trans-elements was undertaken to further determine TF-miRNA regulatory relations, which are necessary to unravel the transcriptional regulation of miRNA genes. Based on miRNA and annotated gene expression profiles, the term “coTFBS” was introduced to detect common transcription factors and the corresponding binding sites within the promoter regions of each miRNA and its coexpressed annotated genes. The computational pipeline was successfully established to filter redundancy due to short sequence motifs for TFBS pattern search. Eventually, we identified more convinced TF-miRNA regulatory relations for 225 human miRNAs. This valuable information is helpful in understanding miRNA functions and provides knowledge to evaluate the therapeutic potential in clinical research. Once most expression profiles of miRNAs in the latest database are completed, TF candidates of more miRNAs can be explored by this filtering approach in the future.

Combination of microRNA expression profiling with genome-wide SNP genotyping to construct a coronary artery disease-related miRNA-miRNA synergistic network.

In recent years, microRNAs (miRNAs) were found to play critical roles in many important biological processes. On the other hand, the rapid development of genome-wide association studies (GWAS) help identify potential genetic variants associated with the disease phenotypic variance. Therefore, we suggested a combined analysis of microRNA expression profiling with genome-wide Single Nucleotide Polymorphism (SNP) genotyping to identify potential disease-related biomarkers. Considering functional SNPs in miRNA genes or target sites might be important signals associated with human complex diseases, we constructed a miRNA-miRNA synergistic network related to coronary artery disease (CAD) by performing a genome-wide scan for SNPs in human miRNA 3' -untranslated regions (UTRs) target sites and computed potential SNP cooperation effects contributing to disease based on potential miRNA-SNP interactions reported recently. Furthermore, we identified some potential CAD-related miRNAs by analyzing the constructed miRNAmiRNA synergistic network. As a result, the predicted miRNA-miRNA network and miRNA clusters were validated by significantly high interaction effects of CAD-related miRNAs. Accurate classification performances were obtained for all of the identified miRNA clusters, and the sensitivity and specificity were all more than 90%. The network topological analysis confirmed some novel CAD-related miRNAs identified recently by experiments. Our method might help to understand miRNA function and CAD disease, as well as to explore the novel mechanisms involved.