Closing the circle : current state and perspectives of circular RNA databases

Circular RNAs (circRNAs) are covalently closed RNA molecules that have been linked to various diseases, including cancer. However, a precise function and working mechanism are lacking for the larger majority. Following many different experimental and computational approaches to identify circRNAs, multiple circRNA databases were developed as well. Unfortunately, there are several major issues with the current circRNA databases, which substantially hamper progression in the field. First, as the overlap in content is limited, a true reference set of circRNAs is lacking. This results from the low abundance and highly specific expression of circRNAs, and varying sequencing methods, data-analysis pipelines, and circRNA detection tools. A second major issue is the use of ambiguous nomenclature. Thus, redundant or even conflicting names for circRNAs across different databases contribute to the reproducibility crisis. Third, circRNA databases, in essence, rely on the position of the circRNA back-splice junction, whereas alternative splicing could result in circRNAs with different length and sequence. To uniquely identify a circRNA molecule, the full circular sequence is required. Fourth, circRNA databases annotate circRNAs' microRNA binding and protein-coding potential, but these annotations are generally based on presumed circRNA sequences. Finally, several databases are not regularly updated, contain incomplete data or suffer from connectivity issues. In this review, we present a comprehensive overview of the current circRNA databases and their content, features, and usability. In addition to discussing the current issues regarding circRNA databases, we come with important suggestions to streamline further research in this growing field

CircNet: a database of circular RNAs derived from transcriptome sequencing data

Circular RNAs (circRNAs) represent a new type of regulatory noncoding RNA that only recently has been identified and cataloged. Emerging evidence indicates that circRNAs exert a new layer of post-transcriptional regulation of gene expression. In this study, we utilized transcriptome sequencing datasets to systematically identify the expression of circRNAs (including known and newly identified ones by our pipeline) in 464 RNA-seq samples, and then constructed the CircNet database (http://circnet.mbc.nctu.edu.tw/) that provides the following resources: (i) novel circRNAs, (ii) integrated miRNA-target networks, (iii) expression profiles of circRNA isoforms, (iv) genomic annotations of circRNA isoforms (e.g. 282 948 exon positions), and (v) sequences of circRNA isoforms. The CircNet database is to our knowledge the first public database that provides tissue-specific circRNA expression profiles and circRNA–miRNA-gene regulatory networks. It not only extends the most up to date catalog of circRNAs but also provides a thorough expression analysis of both previously reported and novel circRNAs. Furthermore, it generates an integrated regulatory network that illustrates the regulation between circRNAs, miRNAs and genes

Role of the epithelial-mesenchymal transition-related circular RNA, circ-10720, in non-small-cell lung cancer

Background: Circular RNAs (circRNAs) are non-coding RNAs with a circular structure that have recently emerged as important regulators of tumorogenesis. Recently, several circRNAS, including circ-10720 have been related to epithelial-mesenchymal transition (EMT) process. In the present study, we have analyzed the role of circ-10720 in non-small-cell lung cancer (NSCLC) and studied its prognostic relevance in resected stage I-IIIa NSCLC patients. Methods: Circ-10720 expression was analyzed using a custom TaqMan assay in four NSCLC cell lines (HCC44, A549, H23 and H1299) and in the normal immortalized lung cell line BEAS2B. Silencing of circ10720 was performed using two custom siRNAs which were transfected using lipofectamine 2000. Protein levels were evaluated by Western blot and immunofluorescence. Wound healing and invasion assays were performed to evaluate the impact the circRNA on cell motility. Apoptosis was analyzed by evaluation of Caspase 3-7 activity and proliferation by MTS assay. Moreover, the expression levels of the circRNA were studied in 119 resected NSCLC patients. The expression in tumor tissue was correlated with the main clinicopathological characteristics and with time to relapse (TTR). Results: Circ-10720 was overexpressed in HCC44 and A549 and underexpressed in H23 and H1299 NSCLC cell lines in comparison to BEAS2B normal immortalized lung cell line. CircRNA knockdown in the two circ-10720 overexpressing cell lines was associated with a decrease of Vimentin (VIM) and an increase of E-cadherin (CDH1) protein levels, loss of mesenchymal phenotype, and a significant reduction of migration and invasion capacity. After silencing circ-10720, the apoptosis rate increased and the proliferation was significantly reduced. Furthermore, circ-10720 was upregulated in tumor vs. normal tissue from 119 resected NSCLC patients. In the group of patients not receiving adjuvant treatment, those with high levels of circ-10720 had a shorter TTR than those with low levels and emerged as an independent prognostic value in the multivariate analysis. In tumor tissue, circ-10720 levels positively correlated with the EMT gene Twist1 levels. Conclusions: Circ-10720 regulates EMT, apoptosis and proliferation and acts as a biomarker of relapse in NSCLC

New technologies accelerate the exploration of non-coding RNAs in horticultural plants.

Non-coding RNAs (ncRNAs), that is, RNAs not translated into proteins, are crucial regulators of a variety of biological processes in plants. While protein-encoding genes have been relatively well-annotated in sequenced genomes, accounting for a small portion of the genome space in plants, the universe of plant ncRNAs is rapidly expanding. Recent advances in experimental and computational technologies have generated a great momentum for discovery and functional characterization of ncRNAs. Here we summarize the classification and known biological functions of plant ncRNAs, review the application of next-generation sequencing (NGS) technology and ribosome profiling technology to ncRNA discovery in horticultural plants and discuss the application of new technologies, especially the new genome-editing tool clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems, to functional characterization of plant ncRNAs

circ-NOL10 regulated by MTDH/CASC3 inhibits breast cancer progression and metastasis via multiple miRNAs and PDCD4

Circular RNAs (circRNAs) play important roles in carcinogenesis. Here, we investigated the mechanisms and clinical significance of circ-NOL10, a highly repressed circRNA in breast cancer. Subsequently, we also identified RNA-binding proteins (RBPs) that regulate circ-NOL10. Bioinformatics analysis was utilized to predict regulatory RBPs as well as circ-NOL10 downstream microRNAs (miRNAs) and mRNA targets. RNA immunoprecipitation, luciferase assay, fluorescence in situ hybridization, cell proliferation, wound healing, Matrigel invasion, cell apoptosis assays, and a xenograft model were used to investigate the function and mechanisms of circ-NOL10 in vitro and in vivo. The clinical value of circ-NOL10 was evaluated in a large cohort of breast cancer by quantitative real-time PCR. Circ-NOL10 is downregulated in breast cancer and associated with aggressive characteristics and shorter survival time. Upregulation of circ-NOL10 promotes apoptosis, decreases proliferation, and inhibits invasion and migration. Furthermore, circ-NOL10 binds multiple miRNAs to alleviate carcinogenesis by regulating PDCD4. CASC3 and metadherin (MTDH) can bind directly to circ-NOL10 with characterized motifs. Accordingly, ectopic expression or depletion of CASC3 or MTDH leads to circ-NOL10 expression changes, suggesting that these two RBPs modulate circ-NOL10 in cancer cells. circ-NOL10 is a novel biomarker for diagnosis and prognosis in breast cancer. These results highlight the importance of therapeutic targeting of the RBP-noncoding RNA (ncRNA) regulation network

A highly conserved circular RNA is required to keep neural cells in a progenitor state in the mammalian brain

circSLC45A4 is the main RNA splice isoform produced from its genetic locus and one of the highest expressed circRNAs in the developing human frontal cortex. Knockdown of this highly conserved circRNA in a human neuroblastoma cell line is sufficient to induce spontaneous neuronal differentiation, measurable by increased expression of neuronal marker genes. Depletion of circSlc45a4 in the developing mouse cortex causes a significant reduction of the basal progenitor pool and increases the expression of neurogenic regulators. Furthermore, knockdown of circSlc45a4a induces a significant depletion of cells in the cortical plate. In addition, deconvolution of the bulk RNA-seq data with the help of single-cell RNA-seq data validates the depletion of basal progenitors and reveals an increase in Cajal-Retzius cells. In summary, we present a detailed study of a highly conserved circular RNA that is necessary to maintain the pool of neural progenitors in vitro and in vivo

CRIT:Identifying RNA-binding protein regulator in circRNA life cycle via non-negative matrix factorization

Circular RNAs (circRNAs) are endogenous non-coding RNAs that regulate gene expression and participate in carcinogenesis. However, the RNA-binding proteins (RBPs) involved in circRNAs biogenesis and modulation remain largely unclear. We developed the circRNA regulator identification tool (CRIT), a non-negative matrix-factorization-based pipeline to identify regulating RBPs in cancers. CRIT uncovered 73 novel regulators across thousands of samples by effectively leveraging genomics data and functional annotations. We demonstrated that known RBPs involved in circRNA control are significantly enriched in these predictions. Analysis of circRNA-RBP interactions using two large cross-linking immunoprecipitation (CLIP) databases, we validated the consistency between CRIT prediction and the CLIP experiments. Furthermore, newly discovered RBPs are functionally connected with authentic circRNA regulators by various biological associations, such as physical interaction, similar binding motifs, common transcription factor modulation, and co-expression. When analyzing RNA sequencing (RNA-seq) datasets after short hairpin RNA (shRNA)/small interfering RNA (siRNA) knockdown, we found several novel RBPs that can affect global circRNA expression, which strengthens their role in the circRNA life cycle. The above evidence provided independent confirmation that CRIT is a useful tool to capture RBPs in circRNA processing. Finally, we show that authentic regulators are more likely the core splicing proteins and peripheral factors and usually harbor more alterations in the vast majority of cancers

Identification of circular RNAs in porcine sperm and evaluation of their relation to sperm motility

Altres ajuts: CERCA Programme/Generalitat de CatalunyaCircular RNAs (circRNAs) are emerging as a novel class of noncoding RNAs which potential role as gene regulators is quickly gaining interest. circRNAs have been studied in different tissues and cell types across several animal species. However, a thorough characterization of the circRNAome in ejaculated sperm remains unexplored. In this study, we profiled the sperm circRNA catalogue using 40 porcine ejaculates. A complex population of 1,598 circRNAs was shared in at least 30 of the 40 samples. Generally speaking, the predicted circRNAs presented low abundances and were tissue-specific. Around 80% of the circRNAs identified in the boar sperm were reported as novel. Results from abundance correlation between circRNAs and miRNAs together with the prediction of microRNA (miRNA) target sites in circRNAs suggested that circRNAs may act as miRNA sponges. Moreover, we found significant correlations between the abundance of 148 exonic circRNAs and sperm motility parameters. Two of these correlations, involving ssc_circ_1458 and ssc_circ_1321, were confirmed by RT-qPCR using 36 additional samples with extreme and opposite sperm motility values. Our study provides a thorough characterization of circRNAs in sperm and suggests that circRNAs hold potential as noninvasive biomarkers for sperm quality and male fertility

Acfs: accurate circRNA identification and quantification from RNA-Seq data

Circular RNAs (circRNAs) are a group of single-stranded RNAs in closed circular form. They are splicing-generated, widely expressed in various tissues and have functional implications in development and diseases. To facilitate genome-wide characterization of circRNAs using RNA-Seq data, we present a freely available software package named acfs. Acfs allows de novo, accurate and fast identification and abundance quantification of circRNAs from single- and paired-ended RNA-Seq data. On simulated datasets, acfs achieved the highest F1 accuracy and lowest false discovery rate among current state- of-the-art tools. On real-world datasets, acfs efficiently identified more bona fide circRNAs. Furthermore, we demonstrated the power of circRNA analysis on two leukemia datasets. We identified a set of circRNAs that are differentially expressed between AML and APL samples, which might shed light on the potential molecular classification of complex diseases using circRNA profiles. Moreover, chromosomal translocation, as manifested in numerous diseases, could produce not only fusion transcripts but also fusion circRNAs of clinical relevance. Featured with high accuracy, low FDR and the ability to identify fusion circRNAs, we believe that acfs is well suited for a wide spectrum of applications in characterizing the landscape of circRNAs from non- model organisms to cancer biology

Nitroxoline suppresses metastasis in bladder cancer via EGR1/circNDRG1/miR-520h/smad7/EMT signaling pathway

Bladder cancer is one of the most common and deadly cancer worldwide. Current chemotherapy has shown limited efficacy in improving outcomes for patients. Nitroxoline, an old and widely used oral antibiotic, which was known to treat for urinary tract infection for decades. Recent studies suggested that nitroxoline suppressed the tumor progression and metastasis, especially in bladder cancer. However, the underlying mechanism for anti-tumor activity of nitroxoline remains unclear. Methods: CircRNA microarray was used to explore the nitroxoline-mediated circRNA expression profile of bladder cancer lines. Transwell and wound-healing assay were applied to evaluate the capacity of metastasis. ChIP assay was chosen to prove the binding of promotor and transcription factor. RNA-pulldown assay was performed to explore the sponge of circRNA and microRNA. Results: We first identified the circNDRG1 (has_circ_0085656) as a novel candidate circRNA. Transwell and wound-healing assay demonstrated that circNDRG1 inhibited the metastasis of bladder cancer. ChIP assay showed that circNDRG1 was regulated by the transcription factor EGR1 by binding the promotor of host gene NDRG1. RNA-pulldown assay proved that circNDRG1 sponged miR-520h leading to the overexpression of smad7, which was a negative regulatory protein of EMT. Conclusions: Our research revealed that nitroxoline may suppress metastasis in bladder cancer via EGR1/circNDRG1/miR-520h/smad7/EMT signaling pathway