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MicroRNAs (miRNAs) regulate gene expression via transcript degradation and translational inhibition, and they may also function as long distance signaling molecules. Circulatory miRNAs are either protein-bound or packaged within vesicles (exosomes). Ten young men (24.6 ± 4.0 yr) underwent a single bout of high-intensity interval cycling exercise. Vastus lateralis biopsies and plasma were collected immediately before and after exercise, as well as 4 h following the exercise bout. Twenty-nine miRNAs previously reported to be regulated by acute exercise were assessed within muscle, venous plasma, and enriched circulatory exosomes via qRT-PCR. Of the 29 targeted miRNAs, 11 were altered in muscle, 8 in plasma, and 9 in the exosome fraction. Although changes in muscle and plasma expression were bidirectional, all regulated exosomal miRNAs increased following exercise. Three miRNAs were altered in all three sample pools (miR-1-3p, -16-5p, and -222-3p), three in both muscle and plasma (miR-21-5p, -134-3p, and -107), three in both muscle and exosomes (miR-23a-3p, -208a-3p, and -150-5p), and three in both plasma and exosomes (miR-486-5p, -126-3p, and -378a-5p). There was a marked discrepancy between the observed alterations between sample pools. A subset of exosomal miRNAs increased in abundance following exercise, suggesting an exercise-induced release of exosomes enriched in specific miRNAs. The uniqueness of the exosomal miRNA response suggests its relevance as a sample pool that needs to be further explored in better understanding biological functions.

To profile the small RNA cargo carried by trophoblast debris derived from the placenta during normal and preeclamptic pregnancies and to determine whether trophoblast debris can deliver its small RNAs to endothelial cells with functional consequences. We confirmed that trophoblast debris can deliver its small RNAs contents to recipient endothelial cells during the co-culture. Next generation sequencing was employed to profile the small RNA contents in both normotensive and preeclamptic trophoblast debris. We identified 1278 mature miRNAs and 2646 non-miRNA small RNA fragments contained. Differential expression analysis identified 16 miRNAs (including miR-145), 5 tRNA fragments from 3 different tRNAs, 13 snRNA fragments and 85 rRNA fragments that were present in different levels between preeclamptic and normotensive trophoblast debris. We loaded a miR-145 mimic into normotensive trophoblast debris via transfection of placental explants from which the debris was derived and found the miR-145 loaded debris induced transcriptomic changes in endothelial cells similar to those induced by preeclamptic trophoblast debris. Trophoblast debris deported into maternal circulation can deliver its small RNA contents to maternal cells thereby contributing to feto-maternal communication. Small RNAs that are dysregulated in preeclamptic trophoblast debris might contribute to the endothelial cell activation which is a hallmark of preeclampsia.

Background: The aim of this study was to explore the clinical utility of microRNAs (miRNAs) as improved markers of ovarian granulosa cell tumours (GCTs) for cancer diagnosis and prognosis prediction. Current histopathological and genetic markers, such as the presence of a FOXL2 gene mutation to distinguish between the two major subtypes are not wholly accurate and as such novel biomarkers are warranted., Methods: The miRNA expression profiles of five formalin-fixed, paraffin-embedded (FFPE) adult-GCTs and five juvenile-GCTs were assessed using Affymetrix miRNA 3.0 Arrays and compared for differential expression. Ten miRNAs were assessed in an additional 33 FFPE tumours and four normal granulosa cell samples by quantitative RT-PCR, and their expression correlated to clinical information., Results: MicroRNA array found 37 miRNAs as differentially expressed between the two GCT subtypes ( p  < 0.05, fold change ≥2 and among these, miRs -138-5p, -184, -204-5p, -29c-3p, -328-3p and -501-3p were validated by RT-qPCR as differentially expressed between the two GCT subtypes ( p  < 0.05). In addition, the expression of miR-184 was predictive of tumour recurrence in adult-GCTs, specifically for patients diagnosed with stage I and II and stage I only disease ( p  < 0.001 and p  < 0.05, respectively)., Conclusions: This study is the first to report on global miRNA expression profiles of human ovarian GCTs using FFPE tumour samples. We have validated six miRNAs as novel markers for subtype classification in GCTs with low levels of miR-138-5p correlating with early tumour stage. Low miR-184 abundance was correlated with tumour recurrence in early stage adult-GCT patients as a candidate predictive biomarker. Further studies are now needed to confirm the clinical utility of these miRNAs as diagnostic and recurrence markers, and understand their possible roles in the pathogenesis of GCTs.

miRNAs are a well-studied class of non-coding RNAs, predominantly functioning to down-regulate gene expression from messenger RNA (mRNA) in a targeted manner by binding to complementary sequence on the target mRNA. Many miRNAs have been linked to the development of hallmarks of cancer. miRNAs represent valuable therapeutic targets to exploit in the search for novel cancer treatments, due to their ubiquitous expression and their ability to tightly regulate the gene expression of a whole host of genes and pathways in a single hit. The miRNA system may be harnessed for therapeutic use either through replacement of tumour suppressive miRNAs lost in cancer, or through inhibition of oncogenic miRNAs overexpressed in cancer. There is a large body of work investigating optimal systemic and localised delivery strategies, and while miRNA therapeutics show promise, it is clear that further developments to delivery strategies may be required to allow safe translation of miRNAs to the clinic. The information gleaned from miRNA signatures as biomarkers is already proving invaluable in the fight to better understand and treat individual tumours, and there is great promise to the applications of these small, but mighty molecules in the future of cancer therapeutics.

MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression at a posttranscriptional level. Each miRNA controls the expression of multiple messenger RNAs (mRNAs) and their dysregulation has been implicated in multiple cancer phenotypes. While some miRNAs are upregulated, global downregulation of miRNA expression is often the rule in cancer. A multitude of potential mechanisms drive aberrant miRNA expression in cancer; miRNA coding regions can harbour genomic defects including mutations, amplifications or deletions, and some miRNAs are broadly repressed by transcription factors such as Myc or have epigenetic modifications to their promoter regions such as hypermethylation of CpG islands. Additionally, the suppression of components of the miRNA processing machinery has been shown to reduce mature miRNA expression and contribute to the malignant phenotype. Understanding the mechanisms driving miRNA downregulation is important in uncovering the critical and complex role of miRNAs in cancer biology. This review will outline the multiple mechanisms by which cancer cells suppress miRNA expression., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Background: The green bottle fly maggot, Lucilia sericata, is a species with importance in medicine, agriculture and forensics. Improved understanding of this species' biology is of great potential benefit to many research communities. MicroRNAs (miRNA) are a short non-protein coding regulatory RNA, which directly regulate a host of protein coding genes at the translational level. They have been shown to have developmental and tissue specific distributions where they impact directly on gene regulation. In order to improve understanding of the biology of L. sericata maggots we have performed small RNA-sequencing of their secretions and tissue at different developmental stages., Results: We have successfully isolated RNA from the secretions of L. sericata maggots. Illumina small RNA-sequencing of these secretions and the three tissues (crop, salivary gland, gut) revealed that the most common small RNA fragments were derived from ribosomal RNA and transfer RNAs of both insect and bacterial origins. These RNA fragments were highly specific, with the most common tRNAs, such as GlyGCC, predominantly represented by reads derived from the 5' end of the mature maggot tRNA. Each library also had a unique profile of miRNAs with a high abundance of miR-10-5p in the maggot secretions and gut and miR-8 in the food storage organ the crop and salivary glands. The pattern of small RNAs in the bioactive maggot secretions suggests they originate from a combination of saliva, foregut and hindgut tissues. Droplet digital RT-PCR validation of the RNA-sequencing data shows that not only are there differences in the tissue profiles for miRNAs and small RNA fragments but that these are also modulated through developmental stages of the insect., Conclusions: We have identified the small-RNAome of the medicinal maggots L. sericata and shown that there are distinct subsets of miRNAs expressed in specific tissues that also alter during the development of the insect. Furthermore there are very specific RNA fragments derived from other non-coding RNAs present in tissues and in the secretions. This new knowledge has applicability in diverse research fields including wound healing, agriculture and forensics.

Objective: To isolate microRNAs (miRNAs) from mesenteric lymph (ML) and peripheral blood and identify those that change with experimental acute pancreatitis (AP). To assess identified AP-associated miRNAs in patient plasma to evaluate them as clinical biomarkers of AP., Background: miRNAs, small non-protein-coding molecules that regulate gene expression, are present in many biological fluids. They are increasingly interesting as biomarkers of disease and as novel signaling molecules in pathogenesis., Methods: Affymetrix miRNA profiling was performed on ML collected from 3 groups of rats with either mild or moderate taurocholate-induced AP and sham controls. Quantitative reverse transcription-polymerase chain reaction was used to validate selected miRNAs in matched rat lymph and plasma and then measured in patients with mild or moderate AP and in healthy volunteers., Results: Eighty-five miRNAs were detectable in rat ML, and many were abundant in all animals irrespective of the presence of AP. Seven miRNAs, comprising miR-375, -217, -148a, -216a, -122, -214, and -138, were increased in ML from rats with AP (P < 0.01). Their abundance also altered with disease severity. miRNAs miR-217, -375, -122, and -148a were also increased in matched rat plasma samples by quantitative reverse transcription-polymerase chain reaction. In the clinical studies, plasma miR-216a was significantly increased in both mild and moderate AP., Conclusions: This study is the first to demonstrate both the presence of circulating miRNAs in lymph and the alteration of specific miRNAs in AP. Furthermore, these miRNAs alter in rat and human AP plasma and have potential to be explored as novel biomarkers of pancreatitis.

MicroRNAs (MiRs) are small, noncoding RNAs that regulate gene expression posttranscriptionally. In this study, we show that MiR-210 is induced by Oct-2, a key transcriptional mediator of B cell activation. Germline deletion of MiR-210 results in the development of autoantibodies from 5 mo of age. Overexpression of MiR-210 in vivo resulted in cell autonomous expansion of the B1 lineage and impaired fitness of B2 cells. Mice overexpressing MiR-210 exhibited impaired class-switched Ab responses, a finding confirmed in wild-type B cells transfected with a MiR-210 mimic. In vitro studies demonstrated defects in cellular proliferation and cell cycle entry, which were consistent with the transcriptomic analysis demonstrating downregulation of genes involved in cellular proliferation and B cell activation. These findings indicate that Oct-2 induction of MiR-210 provides a novel inhibitory mechanism for the control of B cells and autoantibody production.

Objectives: Despite their distinct biology, granulosa cell tumours (GCTs) are treated similarly to other ovarian tumours. Predominantly expressed in granulosa cells, the transcription factor Forkhead Box L2 (FOXL2) is near absent in juvenile-type GCTs. This research aimed to investigate miRNAs as a mechanism of suppression of FOXL2 expression in juvenile-type GCTs., Methods: The miRNA abundance of two GCT cell lines COV434 and KGN was profiled using Affymetrix miRNA GeneChip arrays. Luciferase assays were used to confirm miRNA binding to the 3'UTR of FOXL2. Identified as promising candidates, the miR-17 miRNA family was targeted for knockdown with a miRNA sponge. Additionally, individual family members miR-17, miR-20b and miR-106a were knocked down using Anti-miR™ inhibitors. Subsequently, FOXL2 expression was analysed using RT-qPCR and Western blotting., Results: The profiling of COV434 and KGN cells revealed unique miRNA signatures, with COV434 expressing miR-17 family miRNAs whilst KGN expressed members of the let-7 miRNA gene family. Luciferase assays confirmed miRNA binding to FOXL2's 3'UTR. Reduction of miR-17 family miRNAs increased FOXL2 mRNA expression, however luciferase assays performed in combination with the sponge suggested this is an indirect effect. As no changes in protein were observed, we propose another miRNA is repressing the translation of FOXL2 mRNA., Conclusion: Through miRNA profiling we have begun to unravel the profiles of GCTs, showing that juvenile and adult derived-cell lines are biologically distinct. By expanding on this discovery we may further elucidate the miRNA-mRNA pathways involved in GCT initiation and progression with potential for novel therapeutics for these cancers., (Copyright © 2012 Elsevier Inc. All rights reserved.)

During axon pathfinding, growth cones commonly show changes in sensitivity to guidance cues that follow a cell-intrinsic timetable. The cellular timer mechanisms that regulate such changes are, however, poorly understood. Here we have investigated microRNAs (miRNAs) in the timing control of sensitivity to the semaphorin Sema3A in Xenopus laevis retinal ganglion cell (RGC) growth cones. A developmental profiling screen identified miR-124 as a candidate timer. Loss of miR-124 delayed the onset of Sema3A sensitivity and concomitant neuropilin-1 (NRP1) receptor expression and caused cell-autonomous pathfinding errors. CoREST, a cofactor of a NRP1 repressor, was newly identified as a target and mediator of miR-124 for this highly specific temporal aspect of RGC growth cone responsiveness. Our findings indicate that miR-124 is important in regulating the intrinsic temporal changes in RGC growth cone sensitivity and suggest that miRNAs may act broadly as linear timers in vertebrate neuronal development.

Efficient experimental strategies are needed to validate computationally predicted microRNA (miRNA) target genes. Here we present a large-scale targeted proteomics approach to validate predicted miRNA targets in Caenorhabditis elegans. Using selected reaction monitoring (SRM), we quantified 161 proteins of interest in extracts from wild-type and let-7 mutant worms. We demonstrate by independent experimental downstream analyses such as genetic interaction, as well as polysomal profiling and luciferase assays, that validation by targeted proteomics substantially enriched for biologically relevant let-7 interactors. For example, we found that the zinc finger protein ZTF-7 was a bona fide let-7 miRNA target. We also validated predicted miR-58 targets, demonstrating that this approach is adaptable to other miRNAs. We propose that targeted mass spectrometry can be applied generally to validate candidate lists generated by computational methods or in large-scale experiments, and that the described strategy should be readily adaptable to other organisms.

Background: The differential expression pattern of microRNAs (miRNAs) during mammary gland development might provide insights into their role in regulating the homeostasis of the mammary epithelium. Our aim was to analyse these regulatory functions by deriving a comprehensive tissue-specific combined miRNA and mRNA expression profile of post-natal mouse mammary gland development.We measured the expression of 318 individual murine miRNAs by bead-based flow-cytometric profiling of whole mouse mammary glands throughout a 16-point developmental time course, including juvenile, puberty, mature virgin, gestation, lactation, and involution stages. In parallel whole-genome mRNA expression data were obtained., Results: One third (n = 102) of all murine miRNAs analysed were detected during mammary gland development. MicroRNAs were represented in seven temporally co-expressed clusters, which were enriched for both miRNAs belonging to the same family and breast cancer-associated miRNAs. Global miRNA and mRNA expression was significantly reduced during lactation and the early stages of involution after weaning. For most detected miRNA families we did not observe systematic changes in the expression of predicted targets. For miRNA families whose targets did show changes, we observed inverse patterns of miRNA and target expression. The data sets are made publicly available and the combined expression profiles represent an important community resource for mammary gland biology research., Conclusion: MicroRNAs were expressed in likely co-regulated clusters during mammary gland development. Breast cancer-associated miRNAs were significantly enriched in these clusters. The mechanism and functional consequences of this miRNA co-regulation provide new avenues for research into mammary gland biology and generate candidates for functional validation.

Introduction: The use of cultured cell lines as model systems for normal tissue is limited by the molecular alterations accompanying the immortalisation process, including changes in the mRNA and microRNA (miRNA) repertoire. Therefore, identification of cell lines with normal-like expression profiles is of paramount importance in studies of normal gene regulation., Methods: The mRNA and miRNA expression profiles of several breast cell lines of cancerous or normal origin were measured using printed slide arrays, Luminex bead arrays, and real-time reverse transcription-polymerase chain reaction., Results: We demonstrate that the mRNA expression profiles of two breast cell lines are similar to that of normal breast tissue: HB4a, immortalised normal breast epithelium, and PMC42, a breast cancer cell line that retains progenitor pluripotency allowing in-culture differentiation to both secretory and myoepithelial fates. In contrast, only PMC42 exhibits a normal-like miRNA expression profile. We identified a group of miRNAs that are highly expressed in normal breast tissue and PMC42 but are lost in all other cancerous and normal-origin breast cell lines and observed a similar loss in immortalised lymphoblastoid cell lines compared with healthy uncultured B cells. Moreover, like tumour suppressor genes, these miRNAs are lost in a variety of tumours. We show that the mechanism leading to the loss of these miRNAs in breast cancer cell lines has genomic, transcriptional, and post-transcriptional components., Conclusion: We propose that, despite its neoplastic origin, PMC42 is an excellent molecular model for normal breast epithelium, providing a unique tool to study breast differentiation and the function of key miRNAs that are typically lost in cancer.

MicroRNAs (miRNAs) are causing tremendous excitement in cancer research. MiRNAs are a large class of short non-coding RNAs that are found in many plants, animals and DNA viruses and often act to inhibit gene expression post-transcriptionally. Approximately 500 miRNA genes have been identified in the human genome. Their function is largely unknown, but data from worms, flies, fish and mice suggest that they have important roles in animal growth, development, homeostasis and disease. MiRNA expression profiles demonstrate that many miRNAs are deregulated in human cancers. MiRNAs have been shown to regulate oncogenes, tumour suppressors and a number of cancer-related genes controlling cell cycle, apoptosis, cell migration and angiogenesis. MiRNAs encoded by the mir-17-92 cluster have oncogenic potential and others may act as tumour suppressors. Some miRNAs and their target sites were found to be mutated in cancer. MiRNAs may have great diagnostic potential for human cancer and even miRNA-based cancer therapies may be on the horizon.

Background: MicroRNAs (miRNAs), a class of short non-coding RNAs found in many plants and animals, often act post-transcriptionally to inhibit gene expression., Results: Here we report the analysis of miRNA expression in 93 primary human breast tumors, using a bead-based flow cytometric miRNA expression profiling method. Of 309 human miRNAs assayed, we identify 133 miRNAs expressed in human breast and breast tumors. We used mRNA expression profiling to classify the breast tumors as luminal A, luminal B, basal-like, HER2+ and normal-like. A number of miRNAs are differentially expressed between these molecular tumor subtypes and individual miRNAs are associated with clinicopathological factors. Furthermore, we find that miRNAs could classify basal versus luminal tumor subtypes in an independent data set. In some cases, changes in miRNA expression correlate with genomic loss or gain; in others, changes in miRNA expression are likely due to changes in primary transcription and or miRNA biogenesis. Finally, the expression of DICER1 and AGO2 is correlated with tumor subtype and may explain some of the changes in miRNA expression observed., Conclusion: This study represents the first integrated analysis of miRNA expression, mRNA expression and genomic changes in human breast cancer and may serve as a basis for functional studies of the role of miRNAs in the etiology of breast cancer. Furthermore, we demonstrate that bead-based flow cytometric miRNA expression profiling might be a suitable platform to classify breast cancer into prognostic molecular subtypes.