Thomas B. Hansen

Natural RNA circles function as efficient microRNA sponges

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that act by direct base pairing to target sites within untranslated regions of messenger RNAs. Recently, miRNA activity has been shown to be affected by the presence of miRNA sponge transcripts, the so-called competing endogenous RNA in humans and target mimicry in plants. We previously identified a highly expressed circular RNA (circRNA) in human and mouse brain. Here we show that this circRNA acts as a miR-7 sponge; we term this circular transcript ciRS-7 (circular RNA sponge for miR-7). ciRS-7 contains more than 70 selectively conserved miRNA target sites, and it is highly and widely associated with Argonaute (AGO) proteins in a miR-7-dependent manner. Although the circRNA is completely resistant to miRNA-mediated target destabilization, it strongly suppresses miR-7 activity, resulting in increased levels of miR-7 targets. In the mouse brain, we observe overlapping co-expression of ciRS-7 and miR-7, particularly in neocortical and hippocampal neurons, suggesting a high degree of endogenous interaction. We further show that the testis-specific circRNA, sex-determining region Y (Sry), serves as a miR-138 sponge, suggesting that miRNA sponge effects achieved by circRNA formation are a general phenomenon. This study serves as the first, to our knowledge, functional analysis of a naturally expressed circRNA.

miRNA‐dependent gene silencing involving Ago2‐mediated cleavage of a circular antisense RNA

MicroRNAs (miRNAs) are ∼22 nt non‐coding RNAs that typically bind to the 3′ UTR of target mRNAs in the cytoplasm, resulting in mRNA destabilization and translational repression. Here, we report that miRNAs can also regulate gene expression by targeting non‐coding antisense transcripts in human cells. Specifically, we show that miR‐671 directs cleavage of a circular antisense transcript of the Cerebellar Degeneration‐Related protein 1 (CDR1) locus in an Ago2‐slicer‐dependent manner. The resulting downregulation of circular antisense has a concomitant decrease in CDR1 mRNA levels, independently of heterochromatin formation. This study provides the first evidence for non‐coding antisense transcripts as functional miRNA targets, and a novel regulatory mechanism involving a positive correlation between mRNA and antisense circular RNA levels.

Coordinated epigenetic repression of the miR-200 family and miR-205 in invasive bladder cancer.

MicroRNAs (miRNA) are small noncoding RNAs commonly deregulated in cancer. The miR‐200 family (miR‐200a, ‐200b, ‐200c, ‐141 and ‐429) and miR‐205 are frequently silenced in advanced cancer and have been implicated in epithelial to mesenchymal transition (EMT) and tumor invasion by targeting the transcriptional repressors of E‐cadherin, ZEB1 and ZEB2. ZEB1 is also known to repress miR‐200c‐141 transcription in a negative feedback loop, but otherwise little is known about the transcriptional regulation of the miR‐200 family and miR‐205. Recently, miR‐200 silencing was also reported in cancer stem cells, implying that miR‐200 deregulation is a key event in multiple levels of tumor biology. However, what prevents miR‐200 expression remains largely unanswered. Here we report concerted transcriptional regulation of the miR‐200 and miR‐205 loci in bladder tumors and bladder cell lines. Using a combination of miRNA expression arrays, qPCR assays and mass spectrometry DNA methylation analyses, we show that the miR‐200 and miR‐205 loci are specifically silenced and gain promoter hypermethylation and repressive chromatin marks in muscle invasive bladder tumors and undifferentiated bladder cell lines. Moreover, we report that miR‐200c expression is significantly correlated with early stage T1 bladder tumor progression, and propose miR‐200 and miR‐205 silencing and DNA hypermethylation as possible prognostic markers in bladder cancer. In addition, we observe that the mesoderm transcription factor TWIST1 and miR‐200 expression are inversely correlated in bladder tumor samples and cell lines. TWIST1 associates directly with the miR‐200 and miR‐205 promoters, and may act as a repressor of miR‐200 and miR‐205 expression.

A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity

The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3′-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity.

Circular RNA and miR-7 in Cancer

MicroRNAs (miRNA) play important roles in fine-tuning gene expression and are often deregulated in cancer. The identification of competing endogenous RNA and circular RNA (circRNA) as important regulators of miRNA activity underscores the increasing complexity of ncRNA-mediated regulatory networks. Particularly, the recently identified circular RNA, ciRS-7, which acts as a designated miR-7 inhibitor/sponge, has conceptually changed the mechanistic understanding of miRNA networks. As miR-7 modulates the expression of several oncogenes, disclosing the regulation of miR-7 activity will likely advance the understanding of various cancer etiologies. Here, we review the current knowledge about the ciRS-7/miR-7 axis in cancer-related pathways and discuss possible models explaining the relevance of coexpressing miR-7 along with a circRNA inhibitor.

A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects.

Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (position 2–8 of either siRNA strand counting from the 5′-end) and complementary sequences in the 3′UTR of (off-) targets. It was previously shown that chemical modification of siRNAs can reduce off-targeting but only very few modifications have been tested leaving more to be identified. Here we developed a luciferase reporter-based assay suitable to monitor siRNA off-targeting in a high throughput manner using stable cell lines. We investigated the impact of chemically modifying single nucleotide positions within the siRNA seed on siRNA function and off-targeting using 10 different types of chemical modifications, three different target sequences and three siRNA concentrations. We found several differently modified siRNAs to exercise reduced off-targeting yet incorporation of the strongly destabilizing unlocked nucleic acid (UNA) modification into position 7 of the siRNA most potently reduced off-targeting for all tested sequences. Notably, such position-specific destabilization of siRNA–target interactions did not significantly reduce siRNA potency and is therefore well suited for future siRNA designs especially for applications in vivo where siRNA concentrations, expectedly, will be low.

Comparison of circular RNA prediction tools

CircRNAs are novel members of the non-coding RNA family. For several decades circRNAs have been known to exist, however only recently the widespread abundance has become appreciated. Annotation of circRNAs depends on sequencing reads spanning the backsplice junction and therefore map as non-linear reads in the genome. Several pipelines have been developed to specifically identify these non-linear reads and consequently predict the landscape of circRNAs based on deep sequencing datasets. Here, we use common RNAseq datasets to scrutinize and compare the output from five different algorithms; circRNA_finder, find_circ, CIRCexplorer, CIRI, and MapSplice and evaluate the levels of bona fide and false positive circRNAs based on RNase R resistance. By this approach, we observe surprisingly dramatic differences between the algorithms specifically regarding the highly expressed circRNAs and the circRNAs derived from proximal splice sites. Collectively, this study emphasizes that circRNA annotation should be handled with care and that several algorithms should ideally be combined to achieve reliable predictions.

The miR-143/-145 cluster regulates plasminogen activator inhibitor-1 in bladder cancer

Background:Upregulation of the proto-oncogene plasminogen activator inhibitor-1 (PAI-1) is a common hallmark of various solid tumours, but the mechanisms controlling its expression are not fully understood.Methods:We investigate microRNAs (miRNAs) regulating PAI-1 in a panel of normal bladder urothelial biopsies, superficial Ta bladder tumours and invasive T1–T4 tumours using expression microarrays and qRT–PCR. The prognostic implications of PAI-1 deregulation are established by tissue microarray staining of non-muscle-invasive bladder tumours. MicroRNA repression of PAI-1 is assayed by ectopic miRNA expression, argonaute immunoprecipitation and luciferase assays.Results:We found that the miR-143/-145 cluster is downregulated in all stages of bladder cancer and inversely correlated with PAI-1 expression. Mature miR-143 and miR-145 are coordinately expressed, and both directly target the PAI-1 3′UTR, leading to reduced PAI-1 mRNA and protein levels. Furthermore, we show that PAI-1 and miR-145 levels may serve as useful prognostic markers for non-muscle-invasive bladder tumours for which accurate progressive outcome is currently difficult to predict.Conclusion:This report provides the first evidence for direct miRNA regulation of PAI-1 in bladder cancer. We also demonstrate mRNA co-targeting by a cluster of non-family miRNAs, and suggest miR-145 and PAI-1 as clinically relevant biomarkers in bladder cancer.

Intracellular siRNA and precursor miRNA trafficking using bioresponsive copolypeptides

Small interfering RNAs (siRNAs) can induce specific gene silencing through cytoplasmic mRNA cleavage and nuclear transcriptional silencing, necessitating delivery to different cellular compartments. This study presents a reducible copolypeptide (rCPP) carrier containing different molar ratios of a histidine‐rich peptide (HRP) and nuclear localization sequence (NLS) peptide to modulate intracellular trafficking of transfected siRNA and primary RNA transcripts (pri‐miRNA).

Circular RNAs in cancer: opportunities and challenges in the field

Circular RNA (circRNA) is a novel member of the noncoding cancer genome with distinct properties and diverse cellular functions, which is being explored at a steadily increasing pace. The list of endogenous circRNAs involved in cancer continues to grow; however, the functional relevance of the vast majority is yet to be discovered. In general, circRNAs are exceptionally stable molecules and some have been shown to function as efficient microRNA sponges with gene-regulatory potential. Many circRNAs are highly conserved and have tissue-specific expression patterns, which often do not correlate well with host gene expression. Here we review the current knowledge on circRNAs in relation to their implications in tumorigenesis as well as their potential as diagnostic and prognostic biomarkers and as possible therapeutic targets in future personalized medicine. Finally, we discuss future directions for circRNA cancer research and current caveats, which must be addressed to facilitate the translation of basic circRNA research into clinical use.