Global identification of mRNA-interacting circular RNAs by CLiPPR-Seq.

Although the functional role of circular RNA (circRNA) interaction with microRNAs and proteins has been studied extensively, circRNA interactions with the protein-coding mRNAs in intact cells remain largely unknown. Here, by employing AMT-mediated proximity ligation of RNA-RNA duplexes followed by circRNA enrichment and deep sequencing, we report a novel Cross-Linking Poly(A) Pulldown RNase R Sequencing (CLiPPR-seq) technology which identified hundreds of mRNA-interacting circRNAs in three different cell types, including ßTC6, C2C12 and HeLa cells. Furthermore, CLiPP-seq without RNase R treatment was also performed to identify the mRNA expression in these cells. BLAST analysis of circRNAs in CLiPPR-seq sample with the mRNAs in CLiPP-seq samples determined their potential complementary sequences for circRNA-mRNA interaction. Pulldown of circRNAs and poly(A) RNAs confirmed the direct interaction of circRNAs with target mRNAs. Silencing of mRNA-interacting circRNAs led to the altered expression of target mRNAs in ßTC6 cells, suggesting the role of direct interaction of circRNAs with mRNAs in gene expression regulation. CLiPPR-seq thus represents a novel method for illuminating the myriad of uncharacterized circRNA-mRNA hybrids that may regulate gene expression.

Comparative analysis of circular RNA enrichment methods.

The circRNAs sequencing results vary due to the different enrichment methods and their performance is needed to systematic comparison. This study investigated the effects of different circRNA enrichment methods on sequencing results, including abundance and species of circRNAs, as well as the sensitivity and precision. This experiment was carried out by following four common circRNA enrichment methods: including ribosomal RNA depletion (rRNA-), polyadenylation and poly (A+) RNA depletion followed by RNase R treatment (polyA+RNase R), rRNA-+polyA+RNase R and polyA+RNase R+ rRNA-. The results showed that polyA+RNase R+ rRNA - enrichment method obtained more circRNA number, higher sensitivity and abundance among them; polyA+RNase R method obtained higher precision. The linear RNAs can be thoroughly removed in all enrichment methods except rRNA depletion method. Overall, our results helps researchers to quickly selection a circRNA enrichment of suitable for own study among many enrichment methods, and it provides a benchmark framework for future improvements circRNA enrichment methods.[Figure: see text].

Production of Circular Recombinant RNA in Escherichia coli Using Viroid Scaffolds.

Viroids are small circular, noncoding, highly base-paired RNAs able to infect higher plants. Recently, it has been shown that viroids can be used as very stable scaffolds to produce recombinant RNA in Escherichia coli. Coexpression of an RNA precursor consisting of a viroid monomer, in which the RNA of interest is inserted, flanked by domains of the viroid hammerhead ribozyme, along with a host plant tRNA ligase, the enzyme that catalyzes viroid circularization in infected plants, allows for accumulation of large amounts of the chimeric viroid-RNA of interest in E. coli. Since viroids do not replicate in E. coli, high accumulation most probably results from viroid scaffold stability, resistance to exonucleases due to circularity, and accumulation as a ribonucleoprotein complex with tRNA ligase. Purification of the recombinant RNA from total E. coli RNA is also facilitated by the circular structure of the product.

Exosomal circRNAs contribute to intestinal development via the VEGF signalling pathway in human term and preterm colostrum.

Human breast milk (HBM) provides essential nutrients for newborn growth and development, and contains a variety of biologically active ingredients that can affect gastrointestinal tract and immune system development in breastfed infants. HBM also contains mRNAs, microRNAs and lncRNAs, most of which are encapsulated in milk-derived exosomes and exhibit various important infant development related biological functions. While previous studies have shown that exosomal circRNAs are involved in the intestinal epithelial cells' proliferation and repair. However, the effect of HBM exosomal circRNAs on intestinal development is not clear. In this study, we identified 6756 circRNAs both in preterm colostrum (PC) and term colostrum (TC), of which 66 were upregulated, and 42 were downregulated (|fold change>2|, p < 0.05) in PC. Pathway analysis showed that the VEGF signalling pathway was involved, and network analysis revealed that the differentially expressed circRNAs bound various miRNAs. Further analyses showed that has_circRNA_405708 and has_circRNA_104707 were involved in the VEGF signalling pathway, and that they all bound various mirRNAs. Exosomes found in preterm colostrum (PC) and term colostrum (TC) promoted VEGF protein expression and induced the proliferation and migration of small intestinal epithelial cells (FHCs). Exosomal circRNAs found in human colostrum (HC) binding to related miRNAs may regulate VEGF signalling, and intestinal development.

Novel circRNA discovery in sheep shows evidence of high backsplice junction conservation.

Circular RNAs (circRNAs) are covalently closed circular non-coding RNAs. Due to their structure, circRNAs are more stable and have longer half-lives than linear RNAs making them good candidates for disease biomarkers. Despite the scientific relevance of these molecules, the study of circRNAs in non-model organisms is still in its infancy. Here, we analyse total RNA-seq data to identify circRNAs in sheep from peripheral blood mononuclear cells (PBMCs) and parietal lobe cortex. Out of 2510 and 3403 circRNAs detected in parietal lobe cortex and in PBMCs, a total of 1379 novel circRNAs were discovered. Remarkably, around 63% of all detected circRNAs were found to be completely homologous to a circRNA annotated in human. Functional enrichment analysis was conducted for both tissues based on GO terms and KEGG pathways. The enriched terms suggest an important role of circRNAs from encephalon in synaptic functions and the involvement of circRNAs from PBMCs in basic immune system functions. In addition to this, we investigated the role of circRNAs in repetitive vaccination experiments via differential expression analysis and did not detect any significant relationship. At last, our results support both the miRNA sponge and the miRNA shuttle functions of CDR1-AS in sheep brain. To our knowledge, this is the first study on circRNA annotation in sheep PBMCs or parietal lobe cortex samples.

Genomic analysis of circular RNAs in heart.

BACKGROUND: Heart failure is a leading cause of human morbidity and mortality. Circular RNAs (circRNAs) are a newly discovered class of RNA that have been found to have important physiological and pathological roles. In the current study, we de novo analyzed existing whole transcriptome data from 5 normal and 5 dilated cardiomyopathy (DCM) human heart samples and compared the results with circRNAs that have been previously reported in human, mouse and rat hearts. RESULTS: Our analysis identifies a list of cardiac circRNAs that are reliably detected in multiple studies. We have also defined the top 30 most abundant circRNAs in healthy human hearts which include some with previously unrecognized cardiac roles such as circHIPK3_11 and circTULP4_1. We further found that many circRNAs are dysregulated in DCM, particularly transcripts originating from DCM-related gene loci, such as TTN and RYR2. In addition, we predict the potential of cardiac circRNAs to sponge miRNAs that have reported roles in heart disease. We found that circALMS1_6 has the highest potential to bind miR-133, a microRNA that can regulate cardiac remodeling. Interestingly, we detected a novel class of circRNAs, referred to as read-though (rt)-circRNAs which are produced from exons of two different neighboring genes. Specifically, rt-circRNAs from SCAF8 and TIAM2 were observed to be dysregulated in DCM and these rt-circRNAs have the potential to sponge multiple heart disease-related miRNAs. CONCLUSIONS: In summary, this study provides a valuable resource for exploring the function of circRNAs in human heart disease and establishes a functional paradigm for identifying novel circRNAs in other tissues.

Comparative analysis of the down syndrome hippocampal non-coding RNA transcriptomes using a mouse model.

BACKGROUND: Down syndrome (DS), caused by trisomy 21, is the most common human chromosomal disorder. Hippocampal abnormalities have been believed to be responsible for the DS developmental cognitive deficits. Cumulative evidences indicated that non-coding RNAs (ncRNAs) participated in brain development and function. Currently, few was known whether dysregulated ncRNAs existed in DS whether the dysregulated ncRNAs played important pathology roles in DS. OBJECTIVE: The purpose of this study was generating an overview map of the dysregulated ncRNAs in DS, including the microRNA (miRNA), long ncRNA (lncRNA) and circular RNA (circRNAs). DS mouse models are invaluable tools for further mechanism and therapy studies. METHODS: The well-studied DS mouse model Dp(16)1/Yey was used in this study as it contains the trisomy of the whole human chromosome 21 syntenic region on mouse chromosomes 16. Hippocampi were isolated from pups of seven-days-old. Libraries for miRNA, lncRNA and circRNAs were constructed separately, and the next generation sequencing method was utilized. RESULTS: Differentially expressed (DE) miRNAs, lncRNAs and circRNAs were reported. Relative few regulating relationship were found between the DE miRNAs and DE mRNAs. LncRNAs originated from the trisomic regions expressed in clusters, but not all of them were 1.5-fold increased expressed. Dramatic DE circular RNAs were found in the DS hippocampus. The host genes of the DE circRNAs were enriched on functions which were well-known impaired in DS, e.g. long-term-potentiation, glutamatergic synapse, and GABAergic synapse. CONCLUSIONS: We generated the first DS developmental hippocampal ncRNA transcriptome map. This work laid foundations for further investigations on role of ncRNAs in hippocampal functions.

Automated One-Double-Z Pair BaseScope™ for CircRNA In Situ Hybridization.

Circular RNAs (circRNAs) are single-stranded RNA, typically exons, connected head to tail by back-splicing. The functions of circRNAs include binding of microRNA, regulation of transcription, regulation of alternative splicing, and modulation of immune response. As for other RNA transcripts their levels vary during development and may also become deregulated during disease progression. Different from linear RNAs, the circRNAs are not susceptible to traditional exonuclease activity and therefore more stable in tissues and blood. This makes the circRNAs an attractive new group of potential biomarkers. Specific detection of circRNAs in situ is challenged by the need to discriminate bona fide circRNAs from the linear precursor forms and splice variants that contain largely overlapping sequences. Knowing the sequence around the splice junction site makes the branched DNA probe technology, BaseScope, suitable for selective detection of unique circRNAs. Here, we present the automated application of BaseScope with a one-double-Z pair probe set designed for the junction of circHIPK3.

hsa_circ_0121582 inhibits leukemia growth by dampening Wnt/ß-catenin signaling.

PURPOSE: The prognosis of AML patients with chemotherapy is poor, especially those who are insensitive to and resistant to chemotherapy drugs. To clarify the underlying pathogenesis of AML and provide new therapeutic targets for clinical treatment, we explore the role of circRNA in leukemia. METHODS: High-throughput circRNA sequencing analysis was performed in patients with leukemia and healthy donors. RT-qPCR and western blot analysis were used to determine expression of GSK3ß. RNA pull-down assay was used to detect miRNAs pulled down by hsa_circ_0121582. RNA immunoprecipitation assay was performed to evaluate the binding capacity between TET1 and hsa_circ_0121582. RESULTS: A new and highly stable circRNA was found, which was derived from the reverse splicing of GSK3ß exon 1 to exon 7, and hsa_circ_0121582 was down-regulated in leukemia cells. In gain-of-function experiments, the up-regulated hsa_circ_0121582 inhibited the proliferation of leukemia cells in vitro and in vivo. In the cytoplasm, hsa_circ_0121582 could act as a sponge for miR-224, attenuate the inhibiting effect of miR-224 on GSK3ß, and thus up-regulate the expression level of GSK3ß. In addition, hsa_circ_0121582 could bind to GSK3ß promoter in the nucleus, and recruit DNA demethylase TET1 to ensuring the transcription of GSK3ß. The upregulated GSK3ß inhibited the Wnt/ß-catenin signaling pathway, and reduced the aggregation of ß-catenin in the nucleus, thus inhibited the proliferation of leukemia cells. CONCLUSIONS: This study found that hsa_circ_0121582 was involved in the inhibition of tumor proliferation, and the restoration of hsa_circ_0121582 could be an effective treatment strategy for patients with leukemia.

Screening for differentially expressed circRNA between Kashin-Beck disease and osteoarthritis patients based on circRNA chips.

OBJECTIVE: This research aims to explore differentially expressed circRNA between OA and KBD and potential diagnostic biomarkers. METHODS: Total RNA was extracted from 5 pairs of KBD and OA knee joint cartilage specimens, and the expression of circRNAs was analyzed by Chip Scanning Analysis. The microarray data was verified by quantitative polymerase chain reaction (qRT-PCR). CircRNA-miRNA network was constructed to predict targeting microRNAs of circRNA genes. Peripheral blood samples from 25 KBD patients and 25 OA patients were collected for verification by qRT-PCR. Diagnostic value was evaluated by the area under the receiver operator characteristic (ROC) curve. RESULTS: A total of 1627 circRNAs were differentially expressed between OA and KBD. Five bone and joint disease-related circRNAs were chosen for qRT-PCR validation. The difference in expression profile of hsa_circRNA_0020014 was confirmed by qRT-PCR, and its circRNA-miRNA regulation network was set up. The ROC curve demonstrated that hsa_circ_0020014_CBC1 in peripheral blood could distinguish patients with KBD and OA. CONCLUSION: The expression profiles of circRNA were significantly different between OA and KBD. hsa_circRNA_0020014 is a potential biomarker for differential diagnosis between these two diseases.

Identification of Circular RNAs Regulating Islet ß-Cell Autophagy in Type 2 Diabetes Mellitus.

This study is to identify the circular RNA (circRNA) expression profile that is functionally related to pancreatic islet ß-cell autophagy and their potential regulation mechanisms in type 2 diabetes mellitus (T2DM). T2DM rat model was constructed by administration of high-fat and high-sugar diet. ß-cells were isolated from islets by flow cytometry. CircRNA expression profile in ß-cells was detected by circRNA microarrays, and the differentially expressed circRNAs were identified and validated by qRT-PCR. MicroRNA (miRNA) target prediction software and multiple bioinformatic approaches were used to construct a map of circRNA-miRNA interactions for the differentially expressed circRNAs. A total of 825 differentially expressed circular transcripts were identified in T2DM rats compared with control rats, among which 388 were upregulated and 437 were downregulated. Ten circRNAs were identified to have significant differences by qRT-PCR. GO analysis enriched terms such as organelle membrane and protein binding and the top enriched pathways for the circRNAs included MAPK signaling pathway. The differentially expressed circRNAs might involve in MAPK signaling pathway, apoptosis, and Ras signaling pathway. We speculate that these circRNAs, especially rno_circRNA_008565, can regulate the autophagy of islet ß-cells via interactions with miRNA. Dysregulation of several circRNAs may play a role in T2DM development, and rno_circRNA_008565 may be a potential regulator of ß-cell autophagy.

Identifying circRNA-associated-ceRNA networks in retinal neovascularization in mice.

Retinal neovascularization is a complication which caused human vision loss severely. It has been shown that circular RNAs (circRNAs) play essential roles in gene regulation. However, circRNA expression profile and the underlying mechanisms in retinal neovascular diseases remain unclear. In the present study, we identified altered circRNAs in the retinas of oxygen-induced retinopathy (OIR) mouse model by microarray profiling. Microarray analysis revealed that 539 circRNAs were significantly altered in OIR retinas compared with controls. Among them, 185 up-regulated and 354 down-regulated circRNAs were identified. The expression levels of 4 altered circRNAs including mmu_circRNA_002573, mmu_circRNA_011180, mmu_circRNA_016108 and mmu_circRNA_22546 were validated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Bioinformatic analysis with validated circRNAs such as competing endogenous RNA (ceRNA) regulatory networks with Gene Ontology (GO) enrichment analysis demonstrated that qRT-PCR validated circRNAs were associated with cellular process, cell part and phosphoric ester hydrolase activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that MAPK signaling pathway and renin-angiotensin system were related to validated circRNAs, suggesting these pathways may participate in pathological angiogenesis. The results together suggested that circRNAs were aberrantly expressed in OIR retinas and may play potential roles in retinal neovascular diseases.

Preliminary Exploration of hsa_circ_0032131 Levels in Peripheral Blood as a Potential Diagnostic Biomarker of Osteoarthritis.

Background: Osteoarthritis (OA) is a common chronic degenerative joint disease characterized by articular cartilage degeneration and synovitis. CircRNAs are increasingly being recognized as functional endogenous RNAs with a stable structure and high tissue specificity. Recent studies have shown that some circRNAs may be involved in the initiation and progression of OA and that there is differential expression of circRNAs in chondrocytes in vitro isolated from patients with OA. Purpose: In this study, we aimed to determine if circRNA levels in the peripheral blood of Chinese Han patients with OA would be diagnostic based on the previous in vitro studies. Methods: We collected peripheral blood samples from 25 patients suffering from OA and 25 healthy controls and measured hsa_circ_0032131_CBC1 RNA levels through quantitative RT-PCR (qRT-PCR). The statistical basis for evaluating the diagnostic value was to calculate the area under the receiver operator characteristic (ROC) curve. Results: The results of the qRT-PCR for hsa_circ_0032131_CBC1 were consistent with those of the microarray analysis. The ROC curve shows that hsa_circ_0032131 holds diagnostic value for OA (0.8455, p < 0.01). Conclusions: Our research indicates that differentially expressed circRNAs may be involved in the development of OA and could be used diagnostically.

Screening for Differentially Expressed Circular RNAs in the Cartilage of Osteoarthritis Patients for Their Diagnostic Value.

Background: Osteoarthritis (OA) is the most prevalent osteoarticular disease, which typically involves chronic cartilage degeneration and synovitis. The latest research shows that circular RNAs (circRNAs) play a role in the development of a variety of diseases, including osteoarthrosis. Purposes: The aim of this study was to explore the expression of circRNAs in OA chondrocytes and predict biomarkers for diagnosis. Materials and Methods: The circRNA expression profile was analyzed through use of the Gene Spring software V13.0; differentially expressed circRNAs were screened by comparing OA chondrocytes and normal articular chondrocytes. We validated the microarray data by quantitative real-time polymerase chain reaction analyses of OA chondrocytes and chondrocytes from normal controls. TargetScan software and miRanda software were used to predict networks of circRNA-miRNA interactions in cartilage. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analyses were applied to predict the functions of differentially expressed circRNAs. Results: Overall, 1380 circRNAs were differentially expressed between OA chondrocytes and normal articular chondrocytes (fold-change ≥2, p ≤ 0.05), including 215 that were upregulated and 1165 that were downregulated circRNAs. After analyzing the differentially expressed circRNA genes, the top 20 enriched GO entries and KEGG pathways were annotated. The hsa_circrna_0032131 was identified for further analysis. A circRNA-miRNA network was constructed to represent the 10 most likely target genes associated with the validated circRNA. Conclusions: Our research suggests that some of the differentially expressed circRNAs in OA chondrocytes compared to normal chondrocytes are etiologically associated with the pathological process of OA. It was found that hsa_circRNA_0032131 likely participates in the initiation and progression of OA and has potential as a diagnostic marker. Clinical Relevance: To analyze the difference of circRNA expression profiles between OA and normal controls and explore biomarkers for diagnosis.

Identification of circular RNA Hsa_circ_0001879 and Hsa_circ_0004104 as novel biomarkers for coronary artery disease.

BACKGROUND AND AIMS: The role of circular RNAs (circRNAs) in coronary artery disease (CAD) remains elusive. The aim of the present study was to profile circRNAs expression in CAD patients and assess diagnostics biomarkers for CAD. METHODS: The circRNA profiles of 24 CAD patients and 7 controls were assessed by microarray. The expression levels of candidate circRNAs were further verified by qRT-PCR in large cohorts. Logistic regression analysis and receiver operating characteristic were conducted to assess the diagnostic value. Gain-of-function approach was used to determine the functional significance of validated circRNA in THP-1-derived macrophages. RESULTS: A total of 624 circRNAs and 171 circRNAs were significantly upregulated and downregulated, respectively, in CAD patients relative to controls. Hsa_circ_0001879 and hsa_circ_0004104 were validated to be significantly upregulated in large cohorts. The receiver operating characteristics analysis of hsa_circ_0001879 and hsa_circ_0004104 in CAD patients and controls showed that the area under curve was 0.703 (95% confidence interval: 0.656-0.750; p < 0.001) and 0.700 (95% confidence interval: 0.646-0.755; p < 0.001), respectively. The combination of hsa_circ_0001879 and hsa_circ_0004104, together with CAD risk factors, had the better performance to discriminate CAD patients from healthy controls. Overexpression of hsa_circ_0004104 resulted in dysregulation of atherosclerosis-related genes in THP-1-derived macrophages. CONCLUSIONS: We offered a transcriptome-wide overview of aberrantly expressed circRNAs in CAD patients and identified two novel circRNA biomarkers to diagnose CAD. Upregulation of hsa_circ_0004104 might contribute to the pathogenesis of CAD.

Circular RNA expression profile of knee condyle in osteoarthritis by illumina HiSeq platform.

Osteoarthritis (OA) is the utmost commonly arising joint disease. Knee condyles play an essential role during OA progression. Circular RNA (or circRNA) is a novel kind of RNA, which, unlike the well-known linear RNA, plays an important regulatory role in OA on the basis of a previous research. In our study, expression of circRNAs in OA knee condyle was measured by illumine sequencing platform. A total of 197 differentially expressed circRNAs, such as hg38_circ_0007474 and hg38_circ_0000118 were identified, and 21 target miRNAs, 2466 source genes and 166 394 circRNA-miRNA-mRNA pairs were predicted. Further analysis was applied on three OA-related circRNAs (hsa_circ_0045714, hsa_circ_0002485, and hsa_circ_0005567). The results were partly verified by previous studies. Further biological research is needed to unfold the possible pathway and therapeutic target of OA.

FLI1 Exonic Circular RNAs as a Novel Oncogenic Driver to Promote Tumor Metastasis in Small Cell Lung Cancer.

PURPOSE: The aberrantly upregulated Friend leukemia virus integration 1 (FLI1) is closely correlated with the malignant phenotype of small cell lung cancer (SCLC). It is interesting to note that the CRISPR gene knockout by Cas9 gRNAs that target the FLI1 coding region and the posttranscriptional knockdown by shRNAs that target the 3' region of FLI1 mRNA yielded distinct antimetastasis effects in SCLC cells. This study attempts to examine if FLI1 exonic circular RNAs (FECR) function as a new malignant driver that determines the metastatic phenotype in SCLC. EXPERIMENTAL DESIGN: The clinical relevance of FECRs was examined in 56 primary SCLC tissues and 50 non-small cell lung cancer (NSCLC) tissues. The prognostic value of FECRs was examined by measuring serum exosomal FECRs in a longitudinal cohort of patients with SCLC. The oncogenic activity of FECRs was investigated in both SCLC cell lines and animal xenograft studies. Finally, we explored the molecular mechanisms underlying these noncoding RNAs as a malignant driver. RESULTS: Therapeutic comparison of CRISPR Cas9 knockout and shRNA knockdown of FLI1 identified FECRs as a new noncanonical malignant driver in SCLC. Using RNA FISH and quantitative PCR, we found that FECR1 (exons 4-2-3) and FECR2 (exons 5-2-3-4) were aberrantly upregulated in SCLC tissues (P < 0.0001), and was positively associated with lymph node metastasis (P < 0.01). Notably, serum exosomal FECR1 was associated with poor survival (P = 0.038) and clinical response to chemotherapy. Silencing of FECRs significantly inhibited the migration in two highly aggressive SCLC cell lines and reduced tumor metastasis in vivo. Mechanistically, we uncovered that FECRs sequestered and subsequently inactivated tumor suppressor miR584-3p, leading to the activation of the Rho Associated Coiled-Coil Containing Protein Kinase 1 gene (ROCK1). CONCLUSIONS: This study identifies FLI1 exonic circular RNAs as a new oncogenic driver that promotes tumor metastasis through the miR584-ROCK1 pathway. Importantly, serum exosomal FECR1 may serve as a promising biomarker to track disease progression of SCLC.