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Over the last few years, the number of microRNAs in the human genome has become a controversially debated issue. Several publications reported thousands of putative novel microRNAs not included in the curated microRNA gene database MirGeneDB and the repository miRBase. Recently, by using sequencing of ∼300 human tissues and cell lines, the human RNA atlas, an expanded inventory of human RNA annotations, was published, reporting thousands of putative microRNAs. We, the developers of established microRNA prediction tools and hosts of MirGeneDB, raise concerns about the frequently applied prediction and functional validation strategies, briefly discussing the drawbacks of false positive detections. By means of quantifying well-established biogenesis-derived features, we show that the reported novel microRNAs essentially represent false-positives and argue that the human microRNA complement, at about 550 microRNA genes, is already near complete. Output of available tools must be curated as false predictions will misguide scientists looking for biomarkers or therapeutic targets.

High-throughput sequencing for micro-RNAs (miRNAs) to obtain expression estimates is a central method of molecular biology. Surprisingly, there are a number of different approaches to converting sequencing output into micro-RNA counts. Each has their own strengths and biases that impact on the final data that can be obtained from a sequencing run. This chapter serves to make the reader aware of the trade-offs one must consider in analyzing small RNA sequencing data. It then compares two methods, miRge2.0 and the sRNAbench and the steps utilized to output data from their tools.

RNAcentral is a comprehensive database of non-coding RNA (ncRNA) sequences that provides a single access point to 44 RNA resources and >18 million ncRNA sequences from a wide range of organisms and RNA types. RNAcentral now also includes secondary (2D) structure information for >13 million sequences, making RNAcentral the world’s largest RNA 2D structure database. The 2D diagrams are displayed using R2DT, a new 2D structure visualization method that uses consistent, reproducible and recognizable layouts for related RNAs. The sequence similarity search has been updated with a faster interface featuring facets for filtering search results by RNA type, organism, source database or any keyword. This sequence search tool is available as a reusable web component, and has been integrated into several RNAcentral member databases, including Rfam, miRBase and snoDB. To allow for a more fine-grained assignment of RNA types and subtypes, all RNAcentral sequences have been annotated with Sequence Ontology terms. The RNAcentral database continues to grow and provide a central data resource for the RNA community. RNAcentral is freely available at https://rnacentral.org.

Aging is a key risk factor for chronic diseases of the elderly. MicroRNAs regulate post-transcriptional gene silencing through base-pair binding on their target mRNAs. We identified nonlinear changes in age-related microRNAs by analyzing whole blood from 1334 healthy individuals. We observed a larger influence of the age as compared to the sex and provide evidence for a shift to the 5’ mature form of miRNAs in healthy aging. The addition of 3059 diseased patients uncovered pan-disease and disease-specific alterations in aging profiles. Disease biomarker sets for all diseases were different between young and old patients. Computational deconvolution of whole-blood miRNAs into blood cell types suggests that cell intrinsic gene expression changes may impart greater significance than cell abundance changes to the whole blood miRNA profile. Altogether, these data provide a foundation for understanding the relationship between healthy aging and disease, and for the development of age-specific disease biomarkers., Aging is a key risk factor for chronic diseases of the elderly. Here the authors perform large-scale miRNA profiling of blood from individuals of a range of ages and show that common diseases alter the physiological age-related blood microRNA profile.

RNA sequencing data sets rapidly increase in quantity. For microRNAs (miRNAs), frequently dozens to hundreds of billion reads are generated per study. The quantification of annotated miRNAs and the prediction of new miRNAs are leading computational tasks. Now, the increased depth of coverage allows to gain deeper insights into the variability of miRNAs. The analysis of isoforms of miRNAs (isomiRs) is a trending topic, and a range of computational tools for the analysis of isomiRs has been developed. We provide an overview on 27 available computational solutions for the analysis of isomiRs. These include both stand-alone programs (17 tools) and web-based solutions (10 tools) and span a publication time range from 2010 to 2020. Seven of the tools were published in 2019 and 2020, confirming the rising importance of the topic. While most of the analyzed tools work for a broad range of organisms or are completely independent of a reference organism, several tools have been tailored for the analysis of human miRNA data or for plants. While 14 of the tools are general analysis tools of miRNAs, and isomiR analysis is one of their features, the remaining 13 tools have specifically been developed for isomiR analysis. A direct comparison on 20 deep sequencing data sets for selected tools provides insights into the heterogeneity of results. With our work, we provide users a comprehensive overview on the landscape of isomiR analysis tools and in that support the selection of the most appropriate tool for their respective research task.

Trematodes are widespread parasitic flatworms that significantly affect mankind either directly as human parasites, or indirectly via the infection of livestock and the related economic damage. The two most important trematode taxa are the blood flukes Schistosoma and the liver flukes Fasciola, but detection and differentiation of these parasites remains a challenge. Recently, microRNAs (miRNAs) were described from extracellular vesicles (EV) for both parasites secreted into respective hosts. These molecules have been proposed as mediators of parasite-host communication, and potential biomarkers for the detection of parasitic infections from host blood. Our aim here was to study similarities and differences in the miRNA complements of Schistosoma mansoni and Fasciola hepatica, EV-load in particular, to predict their targets and potential functions in the parasite-host interaction. We reanalyzed the known miRNA complements of S. mansoni and F. hepatica and found 16 and 4 previously overlooked, but deeply conserved miRNAs, respectively, further moving their complements closer together. We found distinct miRNA enrichment patterns in EVs both showing high levels of flatworm miRNAs with potential for the detection of an infection from blood. Two miRNAs of the protostome specific MIR-71 and MIR-277 families were highly expressed in EVs and could, therefore, have potential as biomarkers for trematode infection. Curiously, we identified nucleotide differences in the sequence of Mir-277-P2 between S. mansoni and F. hepatica that hold great promise for the distinction of both parasites. To test whether the EV-miRNAs of S. mansoni and F. hepatica could be modulating the expression of host genes, we predicted miRNA targets in 321 human and cattle messenger RNAs that overlapped between both hosts. Of several predicted targets, wnt signaling pathway genes stood out and their suppression likely leads to changes in the glucose concentration in host blood and the reduction of inflammatory and immune responses.

BACKGROUND & AIMS: Chromosomal instability (CIN) is a carcinogenesis event that promotes metastasis and resistance to therapy by unclear mechanisms. Expression of the colon cancer-associated transcript 2 gene (CCAT2), which encodes a long noncoding RNA (lncRNA), associates with CIN, but little is known about how CCAT2 lncRNA regulates this cancer enabling characteristic.METHODS: We performed cytogenetic analysis of colorectal cancer (CRC) cell lines (HCT116, KM12C/SM, and HT29) overexpressing CCAT2 and colon organoids from C57BL/6N mice with the CCAT2 transgene and without (controls). CRC cells were also analyzed by immunofluorescence microscopy, gamma-H2AX, and senescence assays. CCAT2 transgene and control mice were given azoxymethane and dextran sulfate sodium to induce colon tumors. We performed gene expression array and mass spectrometry to detect downstream targets of CCAT2 lncRNA. We characterized interactions between CCAT2 with downstream proteins using MS2 pull-down, RNA immunoprecipitation, and selective 2'-hydroxyl acylation analyzed by primer extension analyses. Downstream proteins were overexpressed in CRC cells and analyzed for CIN. Gene expression levels were measured in CRC and non-tumor tissues from 5 cohorts, comprising more than 900 patients.RESULTS: High expression of CCAT2 induced CIN in CRC cell lines and increased resistance to 5-fluorouracil and oxaliplatin. Mice that expressed the CCAT2 transgene developed chromosome abnormalities, and colon organoids derived from crypt cells of these mice had a higher percentage of chromosome abnormalities compared with organoids from control mice. The transgenic mice given azoxymethane and dextran sulfate sodium developed more and larger colon polyps than control mice given these agents. Microarray analysis and mass spectrometry indicated that expression of CCAT2 increased expression of genes involved in ribosome biogenesis and protein synthesis. CCAT2 lncRNA interacted directly with and stabilized BOP1 ribosomal biogenesis factor (BOP1). CCAT2 also increased expression of MYC, which activated expression of BOP1. Overexpression of BOP1 in CRC cell lines resulted in chromosomal missegregation errors, and increased colony formation, and invasiveness, whereas BOP1 knockdown reduced viability. BOP1 promoted CIN by increasing the active form of aurora kinase B, which regulates chromosomal segregation. BOP1 was overexpressed in polyp tissues from CCAT2 transgenic mice compared with healthy tissue. CCAT2 lncRNA and BOP1 mRNA or protein were all increased in microsatellite stable tumors (characterized by CIN), but not in tumors with microsatellite instability compared with nontumor tissues. Increased levels of CCAT2 lncRNA and BOP1 mRNA correlated with each other and with shorter survival times of patients.CONCLUSIONS: We found that overexpression of CCAT2 in colon cells promotes CIN and carcinogenesis by stabilizing and inducing expression of BOP1 an activator of aurora kinase B. Strategies to target this pathway might be developed for treatment of patients with microsatellite stable colorectal tumors.

A lack of knowledge of the cellular origin of microRNAs (miRNAs) has greatly confounded functional and biomarkers studies. Recently, three studies characterized miRNA expression patterns across >78 human cell types. These combined data expand our knowledge of miRNA expression localization and confirm that many miRNAs show cell-type specific expression patterns.

Since their discovery in 1993, microRNAs (miRNAs) have been identified as important gene regulators in many biological processes and as key molecular players in human disease, including cancer where they show specific pathogenic deregulation. Their remarkable chemical stability, the availability of very sensitive miRNA detection methods and the fact that miRNAs can be extracted from and detected in various kinds of clinically relevant samples, such as solid tissues, body fluids and secretions make them excellent candidate biomarkers. However, no miRNA has yet entered the level of practical clinical relevance. We present a brief background and some key aspects and challenges of miRNAs as cancer biomarkers, we discuss shortfalls and identify possible routes towards the use of miRNAs as reliable biomarkers for cancer.

SUMMARY We have identified and validated a spaceflight-associated microRNA (miRNA) signature that is shared by rodents and humans in response to simulated, short-duration and long-duration spaceflight. Previous studies have identified miRNAs that regulate rodent responses to spaceflight in low-Earth orbit, and we have confirmed the expression of these proposed spaceflight-associated miRNAs in rodents reacting to simulated spaceflight conditions. Moreover, astronaut samples from the NASA Twins Study confirmed these expression signatures in miRNA sequencing, single-cell RNA sequencing (scRNA-seq), and single-cell assay for transposase accessible chromatin (scATAC-seq) data. Additionally, a subset of these miRNAs (miR-125, miR-16, and let-7a) was found to regulate vascular damage caused by simulated deep space radiation. To demonstrate the physiological relevance of key spaceflight-associated miRNAs, we utilized antagomirs to inhibit their expression and successfully rescue simulated deep-space-radiation-mediated damage in human 3D vascular constructs., Graphical Abstract, In Brief Malkani et al. uncover the role of circulating microRNAs as both a potential biomarker for health risks associated with spaceflight and a countermeasure to mitigate the damage caused to the body by the space environment.

Peritoneal malignant mesothelioma is a rare disease with a generally poor prognosis and poor response to chemotherapy. To improve survival there is a need for increased molecular understanding of the disease, including chemotherapy sensitivity and resistance. We here present an unusual case concerning a young woman with extensive peritoneal mesothelioma who had a remarkable response to palliative chemotherapy (platinum/pemetrexed). Tumor samples collected at surgery before and after treatment were analyzed on the genomic and transcriptional levels (exome sequencing, RNA-seq, and smallRNA-seq). Integrative analysis of single nucleotide and copy-number variants, mutational signatures, and gene expression was performed to provide a comprehensive picture of the disease. LATS1/2 were identified as the main mutational drivers together with homozygous loss of BAP1 and PBRM1, which also may have contributed to the extraordinary chemotherapy response. The presence of the S3 mutational signature is consistent with homologous recombination DNA repair defects due to BAP1 loss. Up-regulation of the PI3K/AKT/mTOR pathway after treatment, supported by deactivated PTEN through miRNA regulation, is associated with cancer progression and could explain chemotherapy resistance. The molecular profile suggests potential benefit from experimental targeting of PARP, EZH2, the PI3K/AKT/mTOR pathway and possibly also from immune checkpoint inhibition. In addition to providing the molecular background for this unusual case of peritoneal mesothelioma, the results show the potential value of integrative genomic analysis in precision medicine.

MicroRNAs (miRNAs) regulate the levels of translation of messenger RNAs (mRNAs). At present, the major parameter that can explain the selection of the target mRNA and the efficiency of translation repression is the base pairing between the 'seed' region of the miRNA and its counterpart mRNA

The aim of this study was to identify microRNAs in urinary exosomes that are differently expressed in prostate cancer patients and healthy donors. For this purpose, RNA was extracted from urinary exosomes from 20 prostate cancer patients and 9 healthy males and the microRNAs were analyzed by next generation sequencing. Interestingly, 5 microRNAs – miR-196a-5p, miR-34a-5p, miR-143-3p, miR-501-3p and miR-92a-1-5p – were significantly downregulated in exosomes from prostate cancer patients. Furthermore, RT-qPCR analysis of an independent cohort of 28 prostate cancer patients and 19 healthy males confirmed that miR-196a-5p and miR-501-3p were downregulated in prostate cancer samples. These results suggest that specific microRNAs in urinary exosomes might serve as non-invasive biomarkers for prostate cancer. In particular, miR-196a-5p and miR-501-3p are promising biomarkers that need to be further studied in large patient cohorts. Electronic supplementary material The online version of this article (10.1186/s12943-017-0726-4) contains supplementary material, which is available to authorized users.

Non-coding RNA (ncRNA) molecules have fundamental roles in cells and many are also stable in body fluids as extracellular RNAs. In this study, we used RNA sequencing (RNA-seq) to investigate the profile of small non-coding RNA (sncRNA) in human serum. We analyzed 10 billion lllumina reads from 477 serum samples, included in the Norwegian population-based Janus Serum Bank (JSB). We found that the core serum RNA repertoire includes 258 micro RNAs (miRNA), 441 piwi-interacting RNAs (piRNA), 411 transfer RNAs (tRNA), 24 small nucleolar RNAs (snoRNA), 125 small nuclear RNAs (snRNA) and 123 miscellaneous RNAs (misc-RNA). We also investigated biological and technical variation in expression, and the results suggest that many RNA molecules identified in serum contain signs of biological variation. They are therefore unlikely to be random degradation by-products. In addition, the presence of specific fragments of tRNA, snoRNA, Vault RNA and Y_RNA indicates protection from degradation. Our results suggest that many circulating RNAs in serum can be potential biomarkers.

Background: Recent large-scale undertakings such as ENCODE and Roadmap Epigenomics have generated experimental data mapped to the human reference genome (as genomic tracks) representing a variety of functional elements across a large number of cell types. Despite the high potential value of these publicly available data for a broad variety of investigations, little attention has been given to the analytical methodology necessary for their widespread utilisation. Findings: We here present a first principled treatment of the analysis of collections of genomic tracks. We have developed novel computational and statistical methodology to permit comparative and confirmatory analyses across multiple and disparate data sources. We delineate a set of generic questions that are useful across a broad range of investigations and discuss the implications of choosing different statistical measures and null models. Examples include contrasting analyses across different tissues or diseases. The methodology has been implemented in a comprehensive open-source software system, the GSuite HyperBrowser. To make the functionality accessible to biologists, and to facilitate reproducible analysis, we have also developed a web-based interface providing an expertly guided and customizable way of utilizing the methodology. With this system, many novel biological questions can flexibly be posed and rapidly answered. Conclusions: Through a combination of streamlined data acquisition, interoperable representation of dataset collections, and customizable statistical analysis with guided setup and interpretation, the GSuite HyperBrowser represents a first comprehensive solution for integrative analysis of track collections across the genome and epigenome. The software is available at: https://hyperbrowser.uio.no., This work was supported by the Research Council of Norway (under grant agreements 221580, 218241, and 231217/F20), by the Norwegian Cancer Society (under grant agreements 71220’PR-2006-0433 and 3485238-2013), and by the South-Eastern Norway Regional Health Authority (under grant agreement 2014041).

Summary Liver flukes represent a paraphyletic group of endoparasitic flatworms that significantly affect man either indirectly due to economic damage on livestock or directly as pathogens. A range of studies have focussed on how these macroscopic organisms can evade the immune-system and live inside a hostile environment such as the mammalian liver and bile-ducts. Recently, microRNAs, a class of short non-coding gene-regulators, have been proposed as likely candidates to play roles in this scenario. MicroRNAs (miRNAs) are key players in development and pathogenicity and are highly conserved between metazoans: identical miRNAs can be found in flatworms and mammalians. Interestingly, miRNAs are enriched in extracellular vesicles (EVs) which are secreted by most cells. EVs constitute an important mode of parasite/host interaction and recent data illustrates that miRNAs play a vital part. We have demonstrated the presence of miRNAs in the EVs of the trematode species Dicrocoelium dendriticum and Fasciola hepatica (Fhe), and identified potential immune-regulatory miRNAs with targets in the host. After our initial identification of miRNAs expressed by F. hepatica, an assembled genome and additional miRNA data became available. This has enabled us to update the known complement of miRNAs in EVs and speculate on potential immune-regulatory functions, that we review here. This article is protected by copyright. All rights reserved.

The advent of next-generation sequencing methods is fuelling the discovery of multiple non-coding RNA transcripts with direct implication in cell biology and homeostasis. This new layer of biological regulation seems to be of particular importance in human pathogenesis, including cancer. The aberrant expression of ncRNAs is a feature of prostate cancer, as they promote tumor-suppressive or oncogenic activities, controlling multicellular events leading to carcinogenesis and tumor progression. From the small RNAs involved in the RNAi pathway to the long non-coding RNAs controlling chromatin remodeling, alternative splicing, and DNA repair, the non-coding transcriptome represents the significant majority of transcriptional output. As such, ncRNAs appear as exciting new diagnostic, prognostic, and therapeutic tools. However, additional work is required to characterize the RNA species, their functions, and their applicability to clinical practice in oncology. In this review, we summarize the most important features of ncRNA biology, emphasizing its relevance in prostate carcinogenesis and its potential for clinical applications.

Robust markers of invasiveness may help reduce the overtreatment of in situ carcinomas. Breast cancer is a heterogeneous disease and biological mechanisms for carcinogenesis vary between subtypes. Stratification by subtype is therefore necessary to identify relevant and robust signatures of invasive disease. We have identified microRNA (miRNA) alterations during breast cancer progression in two separate datasets and used stratification and external validation to strengthen the findings. We analyzed two separate datasets (METABRIC and AHUS) consisting of a total of 186 normal breast tissue samples, 18 ductal carcinoma in situ (DCIS) and 1,338 invasive breast carcinomas. Validation in a separate dataset and stratification by molecular subtypes based on immunohistochemistry, PAM50 and integrated cluster classifications were performed. We propose subtype-specific miRNA signatures of invasive carcinoma and a validated signature of DCIS. miRNAs included in the invasive signatures include downregulation of miR-139-5p in aggressive subtypes and upregulation of miR-29c-5p expression in the luminal subtypes. No miRNAs were differentially expressed in the transition from DCIS to invasive carcinomas on the whole, indicating the need for subtype stratification. A total of 27 miRNAs were included in our proposed DCIS signature. Significant alterations of expression included upregulation of miR-21-5p and the miR-200 family and downregulation of let-7 family members in DCIS samples. The signatures proposed here can form the basis for studies exploring DCIS samples with increased invasive potential and serum biomarkers for in situ and invasive breast cancer.

Although microRNAs (miRNAs) are among the most intensively studied molecules of the past 20 years, determining what is and what is not a miRNA has not been straightforward. Here, we present a uniform system for the annotation and nomenclature of miRNA genes. We show that fewer than a third of the 1,881 human miRBase entries, and only approximately 16% of the 7,095 metazoan miRBase entries, are robustly supported as miRNA genes. Furthermore, we show that the human repertoire of miRNAs has been shaped by periods of intense miRNA innovation, and that mature gene products show a very different tempo and mode of sequence evolution than star products. We establish a new open access database -- MirGeneDB (http://mirgenedb.org) -- to catalog this set of robustly supported miRNAs, which complements the efforts of miRBase, but differs from it by annotating the mature versus star products, and by imposing an evolutionary hierarchy upon this curated and consistently named repertoire.

MicroRNAs (miRNAs) are gene regulators that have recently been shown to down-regulate the immune response via extracellular vesicles in the mammalian host of helminthic parasites. Using the miRNA prediction pipeline miRCandRef, we expanded the current miRNA set of the liver fluke Fasciola hepatica (Platyhelminthes, Trematoda) from 16 to 54 miRNAs (42 conserved and 13 novel). Comparing the cellular expression levels with extracellular vesicles, we found all miRNAs expressed and enriched for miRNAs with immuno-regulatory function, tissue growth and cancer. Our findings support the hypothesis that miRNAs are the molecular mediators of the previously demonstrated immune modulatory function of extracellular vesicles. (C) 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.