circRNA Sequencing Research Articles

Hsa_circ_0000264 promotes tumor progression via the hsa-let-7b-5p/HMGA2 axis in head and neck squamous cell carcinoma.

Circular RNAs (CircRNAs) are involved in various tumors. However, their role in head and neck squamous cell carcinoma (HNSCC) is unknown. CircRNA sequencing data showed that hsa_circ_0000264 is significantly upregulated in HNSCC tissues. In this study, we aimed to investigate the role of hsa_circ_0000264 in HNSCC and elucidate its underlying regulation mechanism. RNase R treatment was performed to confirm the loop structure of hsa_circ_0000264. Fluorescence in situ hybridization was performed to show the subcellular localization of hsa_circ_0000264. We then performed wound healing assay, Transwell assay, Western blot, and in vivo experiments to determine the effect of alterations in hsa_circ_0000264 expression. We performed RNA pull-down and dual luciferase reporter assay to identify and confirm the binding sites in RNAs. hsa_circ_0000264 was upregulated in HNSCC tissues and cells, and its loop structure was confirmed. Knockdown of hsa_circ_0000264 inhibited the migration, invasion, and epithelial-to-mesenchymal transition of HNSCC cells in vivo and in vitro. Mechanistically, hsa_circ_000026 upregulation can upregulate the expression of high mobility group AT-hook 2 (HMGA2) by sponging hsa-let-7b-5p, which in turn promotes HNSCC progression. Our results showed that hsa_circ_0000264 promotes HNSCC progression via the hsa-let-7b-5p/HMGA2 axis, and hsa_circ_0000264 can serve as a potential target for HNSCC treatment.

Pro-Inflammatory Cytokines Promote the Transcription of Circular RNAs in Human Pancreatic β Cells.

Circular RNAs (circRNAs) have recently been implicated in impaired β-cell function in diabetes. Using microarray-based profiling of circRNAs in human EndoC-βH1 cells treated with pro-inflammatory cytokines, this study aimed to investigate the expression and possible regulatory roles of circRNAs in human β cells. We identified ~5000 β-cell-expressed circRNAs, of which 84 were differentially expressed (DE) after cytokine exposure. Pathway analysis of the host genes of the DE circRNAs revealed the enrichment of cytokine signaling pathways, indicative of circRNA transcription from inflammatory genes in response to cytokines. Multiple binding sites for β-cell-enriched microRNAs and RNA-binding proteins were observed for the highly upregulated circRNAs, supporting their function as ‘sponges’ or ‘decoys’. We also present evidence for circRNA sequence conservation in multiple species, the presence of cytokine-induced regulatory elements, and putative protein-coding potential for the DE circRNAs. This study highlights the complex regulatory potential of circRNAs, which may play a crucial role during immune-mediated β-cell destruction in type 1 diabetes.

Identification of a dysregulated CircRNA-associated gene signature for predicting prognosis, immune landscape, and drug candidates in bladder cancer.

Increasing evidences have demonstrated that circular RNA (circRNAs) plays a an essential regulatory role in initiation, progression and immunotherapy resistance of various cancers. However, circRNAs have rarely been studied in bladder cancer (BCa). The purpose of this research is to explore new circRNAs and their potential mechanisms in BCa. A novel ceRNA-regulated network, including 87 differentially expressed circRNAs (DE-circRNAs), 126 DE-miRNAs, and 217 DE-mRNAs was constructed to better understanding the biological processes using Cytoscape 3.7.1 based on our previously high-throughput circRNA sequencing and five GEO datasets. Subsequently, five randomly selected circRNAs (upregulated circ_0001681; downregulated circ_0000643, circ_0001798, circ_0006117 and circ_0067900) in 20 pairs of BCa and paracancerous tissues were confirmed using qRT-PCR. Functional analysis results determined that 772 GO functions and 32 KEGG pathways were enriched in the ceRNA network. Ten genes (PFKFB4, EDNRA, GSN, GAS1, PAPPA, DTL, TGFBI, PRSS8, RGS1 and TCF4) were selected for signature construction among the ceRNA network. The Human Protein Atlas (HPA) expression of these genes were consistent with the above sequencing data. Notably, the model was validated in multiple external datasets (GSE13507, GSE31684, GSE48075, IMvigor210 and GSE32894). The immune-infiltration was evaluated by 7 published algorithms (i.e., TIMER, CIBERSORT, CIBERSORT-ABS, QUANTISEQ, MCPCOUNTER, XCELL and EPIC). Next, Correlations between riskscore or risk groups and clinicopathological data, overall survival, recognized immunoregulatory cells or common chemotherapeutic agents of BCa patients were performed using wilcox rank test, chi-square test, cox regression and spearman’s correlation analysis; and, these results are significant. According to R package “GSVA” and “clusterProfiler”, the most significantly enriched HALLMARK and KEGG pathway was separately the ‘Epithelial Mesenchymal Transition’ and ‘Ecm Receptor Interaction’ in the high- vs. low-risk group. Additionally, the functional experiments in vitro also revealed that the overexpression of has_circ_0067900 significantly impaired the proliferation, migration, and invasion capacities of BCa cells. Collectively, the results of the current study provide a novel landscape of circRNA-associated ceRNA-regulated network in BCa. The ceRNA-associated gene model which was constructed presented a high predictive performance for the prognosis, immunotherapeutic responsiveness, and chemotherapeutic sensitivity of BCa. And, has_circ_0067900 was originally proposed as tumor suppressor for patients with BCa.

NSECDA: Natural Semantic Enhancement for CircRNA-Disease Association Prediction.

Increasing evidence suggest that circRNA, as one of the most promising emerging biomarkers, has a very close relationship with diseases. Exploring the relationship between circRNA and diseases can provide novel perspective for diseases diagnosis and pathogenesis. The existing circRNA-disease association (CDA) prediction models, however, generally treat the data attributes equally, do not pay special attention to the attributes with more significant influence, and do not make full use of the correlation and symbiosis between attributes to dig into the latent semantic information of the data. Therefore, in response to the above problems, this paper proposes a natural semantic enhancement method NSECDA to predict CDA. In practical terms, we first recognize the circRNA sequence as a biological language, and analyze its natural semantic properties through the natural language understanding theory; then integrate it with disease attributes, circRNA and disease Gaussian Interaction Profile (GIP) kernel attributes, and use Graph Attention Network (GAT) to focus on the influential attributes, so as to mine the deeply hidden features; finally, the Rotation Forest (RoF) classifier was used to accurately determine CDA. In the gold standard data set CircR2Disease, NSECDA achieved 92.49% accuracy with 0.9225 AUC score. In comparison with the non-natural semantic enhancement model and other classifier models, NSECDA also shows competitive performance. Additionally, 25 of the CDA pairs with unknown associations in the top 30 prediction scores of NSECDA have been proven by newly reported studies. These achievements suggest that NSECDA is an effective model to predict CDA, which can provide credible candidate for subsequent wet experiments, thus significantly reducing the scope of investigations.

CircMERTK modulates the suppressive capacity of tumor-associated macrophage via targeting IL-10 in colorectal cancer.

Macrophages represent the major population in the tumor microenvironment (TME). Recent studies have demonstrated circular RNAs (circRNAs) are implicated in the development and progression of different immune responses and immune diseases. However, the role of circRNAs in the development of tumor-associated macrophages (TAM) remains unknown. Here, we used the circRNA sequencing to identify the differentially expressed circRNAs (DEcircRNAs) in TAM-like cell induced by culture medium of colorectal cancer cell lines. Of note, the expression of circMERTK was remarkably overexpressed in TAMs. The ISH assay displayed that the expressions of circMERTK were mainly overlapped with macrophages marker CD68, and the abundance of circMERTK in CRC tissues was much higher than that in matched normal tissues. Functionally, circMERTK knockdown resulted in attenuated CD8+ T cell apoptosis in the co-culture assay, indicating that circMERTK could have an impact on the immunosuppressive activity of TAM-like cell. Mechanically, TAM-like cell could exert immunosuppressive activity via circMERTK/miR-125a-3p/IL-10 axis. These data suggested that circMERTK could play an important role in TAM activation, and may serve as a potential therapeutic target for CRC.

Expression profiling and functional analysis of circular RNAs in vitro model of intermittent hypoxia-induced liver injury.

Intermittent hypoxia (IH) is a prominent feature of obstructive sleep apnea (OSA) which is increasingly recognized as a key risk factor for liver injury. Circular RNAs (circRNAs) has been suggested to act as a regulator of multiple biological processes. However, there is no study evaluating circRNAs alterations and potential role of circRNAs in OSA-related liver injury. The present study aimed to investigate circRNA expression profiles in vitro model of IH-induced liver injury, as well as potential functional characterization of the differentially expressed circRNAs (DE circRNAs). BRL-3A cells were exposed to IH or normoxia. Cell apoptosis and cell viability were evaluated using flow cytometry and cell counting kit-8, respectively. The expression profile of circRNAs was depicted by circRNA sequencing. The selected circRNAs were verified by quantitative real-time PCR (qRT-PCR). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analyses were employed to predict DE circRNAs functions. The circRNA-miRNA-mRNA regulatory network was constructed. IH treatment caused cell injury in BRL-3A cells. 98 circRNAs were identified as being dysregulated in IH-treated BRL-3A cells. Among them, 58 were up-regulated and 40 were down-regulated. Go and KEGG analyses suggested that the DE circRNAs were predominantly enriched in the biological process such as positive regulation of NF−kappaB transcription factor activity and pathways such as circadian entrainment, Wnt signaling pathway, MAPK signaling pathway, and protein export. 3 up-regulated circRNAs and 3 down-regulated circRNAs with high number of back-splicing sites were chosen for qRT-PCR validation and were consistent with the sequencing data. CircRNA1056 and circRNA805 were predicted to interact with microRNAs that might thereby regulate downstream genes. The study characterized a profile of dysregulated circRNAs in IH-induced BRL-3A cell injury. DE circRNAs may play vital roles in the pathophysiology of IH-induced liver injury. Our findings provide preliminary support for further research in mechanisms and a new theory for the pathogenesis of OSA-related liver injury.

A machine learning framework based on multi-source feature fusion for circRNA-disease association prediction.

Circular RNAs (circRNAs) are involved in the regulatory mechanisms of multiple complex diseases, and the identification of their associations is critical to the diagnosis and treatment of diseases. In recent years, many computational methods have been designed to predict circRNA-disease associations. However, most of the existing methods rely on single correlation data. Here, we propose a machine learning framework for circRNA-disease association prediction, called MLCDA, which effectively fuses multiple sources of heterogeneous information including circRNA sequences and disease ontology. Comprehensive evaluation in the gold standard dataset showed that MLCDA can successfully capture the complex relationships between circRNAs and diseases and accurately predict their potential associations. In addition, the results of case studies on real data show that MLCDA significantly outperforms other existing methods. MLCDA can serve as a useful tool for circRNA-disease association prediction, providing mechanistic insights for disease research and thus facilitating the progress of disease treatment.

A web server for identifying circRNA-RBP variable-length binding sites based on stacked generalization ensemble deep learning network.

Circular RNA (circRNA) can exert biological functions by interacting with RNA-binding protein (RBP), and some deep learning-based methods have been developed to predict RBP binding sites on circRNA. However, most of these methods identify circRNA-RBP binding sites are only based on single data resource and cannot provide exact binding sites, only providing the probability value of a sequence fragment. To solve these problems, we propose a binding sites localization algorithm that fuses binding sites from multiple databases, and further design a stacked generalization ensemble deep learning model named CirRBP to identify RBP binding sites on circRNA. The CirRBP is trained by combining the binding sites from multiple databases and makes predictions by weighted aggregating the predictions of each sub-model. The results show that the CirRBP outperforms any sub-model and existing online prediction model. For better access to our research results, we develop an open-source web application called CRWS (CircRNA-RBP Web Server). Its back-end learning model of the CRWS is a stacked generalization ensemble learning model CirRBP based on different deep learning frameworks. Given a full-length circRNA or fragment sequence and a target RBP, the CRWS can analyze and provide the exact potential binding sites of the target RBP on the given sequence through the binding sites localization algorithm, and visualize it. In addition, the CRWS can discover the most widely distributed motif in each RBP dataset. Up to now, CRWS is the first significant online tool that uses multi-source data to train models and predict exact binding sites. CRWS is now publicly and freely available without login requirement at: http://www.bioinformatics.team.

Identification of circular RNA biomarkers for Pien Tze Huang treatment of CCl4‑induced liver fibrosis using RNA‑sequencing

Pien Tze Huang (PZH), a common hepatoprotective Traditional Chinese Medicine that has been found to be an effective treatment for carbon tetrachloride-induced hepatic damage, including liver fibrosis. Circular RNAs (circRNAs) serve a crucial role in regulating gene expression levels via circRNA/micro (mi)RNA/mRNA networks in several human diseases and biological processes. However, whether circRNAs are involved in the underlying mechanism of the therapeutic effects of PZH on liver fibrosis remains unclear. Therefore, the aim of the present study was to investigate these effects using circRNA expression profiles from PZH-treated fibrotic livers in model mice. A case-control study on >59,476 circRNAs from CCl4-induced (control group, n=6) and PZH-treated (case group, n=6) mice was performed using circRNA sequencing in liver tissues. PZH treatment resulted in the differential expression of 91 circRNAs, including 58 upregulated and 33 downregulated circRNAs. Furthermore, the construction of competing endogenous networks also indicated that differentially expressed circRNAs acted as miRNA sponges. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of miRNA targets demonstrated that PZH-affected circRNAs were mainly involved in biological processes such as ‘positive regulation of fibroblast proliferation’, ‘cellular response to interleukin-1’ and ‘regulation of DNA-templated transcription in response to stress’ and in a number of important pathways, such as ‘TNF signaling pathway’, ‘PI3K-Akt signaling pathway’, ‘IL-17 signaling pathway’ and ‘MAPK signaling pathway’. To further validate the bioinformatics data, reverse transcription–quantitative PCR was performed on seven miRNA targets in a human hepatic stellate LX-2 cell model. The results suggested that seven of the miRNAs exhibited regulatory patterns that were consistent with those of the transcriptome sequencing results. Kaplan-Meier survival analysis demonstrated that the expression levels of dihydrodiol dehydrogenase and solute carrier family 7, member 11 gene were significantly associated with patient survival, 269 patients with liver hepatocellular carcinoma from The Cancer Genome Atlas database. To the best of our knowledge, this was the first study to provide evidence that PZH affects circRNA expression levels, which may serve important roles in PZH-treated fibrotic liver through the regulation of functional gene expression. In conclusion, the present study provided new insights into the mechanism underlying the pathogenesis of liver fibrosis and identified potential novel, efficient, therapeutic targets against liver injury.

Circular RNA circNFKB1 promotes osteoarthritis progression through interacting with ENO1 and sustaining NF-κB signaling

Inflammatory cytokines-induced activation of the nuclear factor κB (NF-κB) pathway plays a critical role in the pathogenesis of osteoarthritis (OA). Circular RNA (circRNA) has been identified as important epigenetic factor in numerous diseases. However, the biological roles of inflammation-related circRNAs in regulating OA pathogenesis remain elusive. Here, we revealed circRNA expression profiles in human primary chondrocytes with interleukin-1β (IL-1β) stimulation by circRNA sequencing. We identified a highly upregulated circRNA, termed as circNFKB1 in inflamed chondrocytes and osteoarthritic cartilage. As a circRNA derived from exon 2–5 of NFKB1, circNFKB1 is located in both cytoplasm and nucleus of chondrocytes. Furthermore, knockdown of circNFKB1 inhibited extracellular matrix (ECM) catabolism and rescued IL-1β impaired ECM anabolism whereas ectopic expression of circNFKB1 significantly promoted chondrocytes degradation in vitro. Moreover, intraarticular injection of adenovirus-circNFKB1 in mouse joints triggered spontaneous cartilage loss and OA development. Mechanistically, circNFKB1 interacted with α-enolase (ENO1), regulated the expression of its parental gene NFKB1 and sustained the activation of NF-κB signaling pathway in chondrocytes. Therefore, this study highlights a novel ENO1-interacting circNFKB1 in OA pathogenesis, and provides valuable insights into understanding the regulatory mechanism of NF-κB signaling in chondrocytes and a promising therapeutic target for the treatment of OA.

High-throughput circular RNA sequencing reveals the profiles of circular RNA in non-cirrhotic hepatocellular carcinoma

BackgroundLiver cirrhosis is a well-known risk factor for hepatocellular carcinoma (HCC). However, some HCC cases can also originate from non-cirrhotic livers. The aim of this study was to identify key circular RNAs (circRNAs) associated with the tumorigenesis of non-cirrhotic liver disease.MethodsThe differently expressed circRNAs between non-cirrhotic and cirrhotic HCCs were assessed with use of high-throughput circRNAs sequencing and validated with quantitative reverse transcription polymerase chain reaction (qRT-PCR). Potential biological functions of these dysregulated circRNAs were predicted with use of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. A circRNA-miRNA-mRNA regulation network was constructed as achieved with use of miRanda software and visualized using Cytoscape software. Biological functions of the four most prominent dysregulated circRNAs identified were confirmed by in vitro experiments. Moreover, possible translations of these dysregulated circRNAs were also predicted.ResultsA total of 393 dysregulated circRNAs were identified between non-cirrhotic and cirrhotic HCC, including 213 that were significantly up-regulated and 180 significantly down-regulated circRNAs. Expression levels of the six most prominent dysregulated circRNAs were further validated using qRT-PCR. Many tumor related miRNAs were involved in the circRNA-miRNA-mRNA networks, including miR-182-5p, miR-561-3p, miR-125a-5p, miR-145, miR-23b-3p and miR-30e-3p, and downstream mRNAs of dysregulated circRNAs were significantly related with biological processes involved in the progression of tumors, including proliferation, migration, differentiation, and focal adhesion. Results from the in vitro experiments demonstrated that the most prominent dysregulated circRNAs exerted notable effects upon the proliferation and migration of HCC cells. Finally, we also identified 19 dysregulated circRNAs having potential for the coding of functional peptides.ConclusionThe results of this present study indicate that circRNAs may play important roles in tumorigenesis of non-cirrhotic HCC. Such findings provide some novel insights and pave the way for the development of future studies directed at investigating the initiation and treatment of HCC.

CircIPO7 Promotes Nasopharyngeal Carcinoma Metastasis and Cisplatin Chemoresistance by Facilitating YBX1 Nuclear Localization.

Cisplatin-based chemotherapy effectively improves the distant-metastasis control in nasopharyngeal carcinoma (NPC), but approximately 30% of patients develop treatment failure due to chemoresistance. However, the underlying mechanisms remain poorly understood. Circular RNA (circRNA) sequencing data were used to identify metastasis-specific circRNAs and the expression of circIPO7 was validated in NPC tissues as well as NPC cell lines by qRT-PCR. The whole transcriptional profile upon circIPO7 knockdown was applied to explore the biological function and regulatory mechanism, which were further confirmed by in vitro and in vivo metastasis/chemosensitivity assays. We also evaluated the value of circIPO7 expression in predicting NPC metastasis and cisplatin chemoresistance by analyzing a cohort of 183 NPC patients. In this study, circIPO7, a novel circRNA, is found to be specifically overexpressed in NPC patients with distant metastasis. Knockdown of circIPO7 in NPC cells suppresses their metastasis and increases sensitivity to cisplatin treatment in vitro and in vivo. Mechanistically, circIPO7 binds to Y-box binding protein-1 (YBX1) protein in the cytoplasm and facilitates its phosphorylation at serine 102 (p-YBX1S102) by the kinase AKT, which further promotes YBX1 nuclear translocation and activates FGFR1, TNC, and NTRK1 transcription. Clinically, higher circIPO7 expression indicates unfavorable distant metastasis-free survival in NPC patients given cisplatin-based chemotherapy. Altogether, this study identifies oncogenic circIPO7 as a prognostic marker after cisplatin-based chemotherapy and as a potential therapeutic target for overcoming metastasis and chemoresistance in NPC.

Immunomodulatory functions of the circ_001678/miRNA-326/ZEB1 axis in non-small cell lung cancer via the regulation of PD-1/PD-L1 pathway.

High-throughput circular RNA (circRNA) sequencing identified circRNA_001678 (circ_001678) as an upregulated circRNA in non-small cell lung cancer (NSCLC) tissues. Hence, the current study sought to investigate the function and the underlying mechanism of circRNA_001678 in immune escape of NSCLC. Briefly, commercially purchased NSCLC cell lines were adopted for in vitro experiment to evaluate the effects of circ_001678 over-expression or knockdown on cell biological functions, including proliferation, migration and invasive abilities. In addition, the effects of circ_001678 on the in vivo tumorigenicity ability were evaluated for verification. Accordingly, we uncovered that circ_001678 over-expression augmented NSCLC progression in vitro and enhanced tumorigenicity ability in vivo. The interaction between circ_001678 and miR-326 predicted online was verified by means of luciferase and RNA pull-down assays. Furthermore, circ_001678 could sponge miR-326 to up-regulate ZEB1. On the other hand, the tumor-promoting effects of circ_001678 could be inhibited by anti-PD-L1/PD-1 treatment. Mechanistically, circ_001678 led to the activation of the PD-1/PD-L1 pathway to promote CD8+ T cell apoptosis, thereby inducing NSCLC cell immune escape via regulation of the miR-326/ZEB1 axis. To conclude, our findings revealed that circ_001678 sponges miR-326 to up-regulate ZEB1 expression and induce the PD-1/PD-L1 pathway-dependent immune escape, thereby promoting the malignant progression of NSCLC.

Vimentin binds to a novel tumor suppressor protein, GSPT1-238aa, encoded by circGSPT1 with a selective encoding priority to halt autophagy in gastric carcinoma

Circular RNAs (circRNAs) are covalently closed, endogenous molecules that are widespread in eukaryotes. Recent evidence indicates that circRNAs play important roles in carcinogenesis. Several circRNAs have been reported to comprise translatable RNA; however, whether circRNAs encode functional proteins remains unknown. In our study, circRNA sequencing was carried out using five pathologically diagnosed gastric carcinoma (GC) samples and their paired adjacent normal tissues, we characterized the circRNA GSPT1 (circGSPT1), which is expressed at low levels in GC. Antibody detections, and mass spectrometry were used to validate active circRNA translation. The spanning junction open reading frame in circGSPT1, driven by an internal ribosome entry site (IRES), encodes a functional peptide, termed GSPT1-238aa. Interestingly, GSPT1-238aa tends to select the start codon used to initiate translation. This is the first finding of selective translation driven by IRES. CircGSPT1 and GSPT1-238aa halted the proliferation, migration, and invasion in GC cells in vitro. We also confirmed that the vimentin/Beclin1/14-3-3 complex interacts with GSPT1-238aa and modulates autophagy via the PI3K/AKT/mTOR signaling pathway in GC cells. Our study reveals that GSPT1-238aa, a novel protein encoded by circGSPT1, halts GC tumorigenesis. We also provide insights into the function and underlying molecular mechanisms of GSPT1-238aa in GC and suggest that this protein represents a novel target for GC treatment.

Prediction of circRNA-Disease Associations Based on the Combination of Multi-Head Graph Attention Network and Graph Convolutional Network.

Circular RNAs (circRNAs) are covalently closed single-stranded RNA molecules, which have many biological functions. Previous experiments have shown that circRNAs are involved in numerous biological processes, especially regulatory functions. It has also been found that circRNAs are associated with complex diseases of human beings. Therefore, predicting the associations of circRNA with disease (called circRNA-disease associations) is useful for disease prevention, diagnosis and treatment. In this work, we propose a novel computational approach called GGCDA based on the Graph Attention Network (GAT) and Graph Convolutional Network (GCN) to predict circRNA-disease associations. Firstly, GGCDA combines circRNA sequence similarity, disease semantic similarity and corresponding Gaussian interaction profile kernel similarity, and then a random walk with restart algorithm (RWR) is used to obtain the preliminary features of circRNA and disease. Secondly, a heterogeneous graph is constructed from the known circRNA-disease association network and the calculated similarity of circRNAs and diseases. Thirdly, the multi-head Graph Attention Network (GAT) is adopted to obtain different weights of circRNA and disease features, and then GCN is employed to aggregate the features of adjacent nodes in the network and the features of the nodes themselves, so as to obtain multi-view circRNA and disease features. Finally, we combined a multi-layer fully connected neural network to predict the associations of circRNAs with diseases. In comparison with state-of-the-art methods, GGCDA can achieve AUC values of 0.9625 and 0.9485 under the results of fivefold cross-validation on two datasets, and AUC of 0.8227 on the independent test set. Case studies further demonstrate that our approach is promising for discovering potential circRNA-disease associations.

CircRNA-3302 promotes endothelial-to-mesenchymal transition via sponging miR-135b-5p to enhance KIT expression in Kawasaki disease

Endothelial-to-mesenchymal transition (EndMT) is implicated in myofibroblast-like cell-mediated damage to coronary artery wall of Kawasaki disease (KD) patients, which subsequently increases the risk of coronary artery aneurysm. Many circular RNAs (circRNAs) have been reported to be associated with cardiovascular diseases. However, the roles and underlying molecular mechanism of circRNAs in KD-associated EndMT remains indefinite. In this research, we screened out circRNA-3302 from human umbilical vein endothelial cells (HUVECs) treated by sera from healthy controls (HCs) or KD patients via circRNA sequencing (circRNA-seq). In addition, circRNA-3302 upregulation was verified in endothelial cells stimulated by KD serum and pathological KD mice modeled with Candida albicans cell wall extracts (CAWS). Moreover, in vitro experiments demonstrated that overexpression of circRNA-3302 could markedly induce EndMT, and silencing of circRNA-3302 significantly alleviated KD serum-mediated EndMT. To further explore the molecular mechanisms of circRNA-3302 inducing EndMT, RNA sequencing (RNA-seq), a dual-luciferase reporter system, nuclear and extra-nuclear RNA isolation, RT-qPCR and Western blot analyses and so on, were utilized. Our data demonstrated that circRNA-3302 contributed to the KD-associated EndMT via sponging miR-135b-5p to enhance KIT expression. Collectively, our results imply that circRNA-3302 plays an important role in KD-associated EndMT, providing new insights into minimizing the risks of developing coronary artery aneurysms.

A circular RNA derived from the insulin receptor locus protects against doxorubicin-induced cardiotoxicity.

Cardiotoxicity leading to heart failure (HF) is a growing problem in many cancer survivors. As specific treatment strategies are not available, RNA discovery pipelines were employed and a new and powerful circular RNA (circRNA)-based therapy was developed for the treatment of doxorubicin-induced HF. The circRNA sequencing was applied and the highly species-conserved circRNA insulin receptor (Circ-INSR) was identified, which participates in HF processes, including those provoked by cardiotoxic anti-cancer treatments. Chemotherapy-provoked cardiotoxicity leads to the down-regulation of Circ-INSR in rodents and patients, which mechanistically contributes to cardiomyocyte cell death, cardiac dysfunction, and mitochondrial damage. In contrast, Circ-INSR overexpression prevented doxorubicin-mediated cardiotoxicity in both rodent and human cardiomyocytes in vitro and in a mouse model of chronic doxorubicin cardiotoxicity. Breast cancer type 1 susceptibility protein (Brca1) was identified as a regulator of Circ-INSR expression. Detailed transcriptomic and proteomic analyses revealed that Circ-INSR regulates apoptotic and metabolic pathways in cardiomyocytes. Circ-INSR physically interacts with the single-stranded DNA-binding protein (SSBP1) mediating its cardioprotective effects under doxorubicin stress. Importantly, in vitro transcribed and circularized Circ-INSR mimics also protected against doxorubicin-induced cardiotoxicity. Circ-INSR is a highly conserved non-coding RNA which is down-regulated during cardiotoxicity and cardiac remodelling. Adeno-associated virus and circRNA mimics-based Circ-INSR overexpression prevent and reverse doxorubicin-mediated cardiomyocyte death and improve cardiac function. The results of this study highlight a novel and translationally important Circ-INSR-based therapeutic approach for doxorubicin-induced cardiac dysfunction.

CircRNA circ_0006156 inhibits the metastasis of prostate cancer by blocking the ubiquitination of S100A9

Circular RNAs (circRNAs) have been demonstrated to play vital roles in cancer development and progression. However, studies on the association between circRNAs and prostate cancer (PCa) are still lacking. CircRNA sequencing of two pairs of PCa tissues and adjacent normal tissues was conducted in the present study, and qRT–PCR was performed to verify the results. Functional experiments were performed to investigate cellular functions after specific changes. Mass spectrometry analysis after RNA pull-down experiments and Co-IP assays were further conducted. Downstream target proteins were predicted via online databases and detected in vitro by Western blot analysis and in vivo by immunohistochemistry. Hsa_circ_0006156 (subsequently named circ_0006156) expresses at low levels in both PCa tissues and cells, and it significantly inhibits the migration and invasion of PCa cells. Circ_0006156 binds to and blocks the ubiquitination of S100A9. Moreover, functional assays revealed that circ_0006156 represses the malignant progression of PCa by binding to S100A9. Finally, in vivo experiments showed that circ_0006156 suppresses PCa migration and invasion by increasing S100A9, revealing circ_0006156 as a potential novel effective target for PCa treatment.

P595: DISCOVERY OF NOVEL CIRCULAR RNAS (CIRCRNAS) OF THE APOPTOSIS-RELATED BAX AND BCL2L12 GENES WITH A MULTIFACETED ROLE IN CHRONIC LYMPHOCYTIC LEUKEMIA, USING CUTTING-EDGE NANOPORE SEQUENCING TECHNOLOGY

Background: Circular RNAs (circRNAs), a novel RNA type generated by back-splicing, are key indirect regulators of gene expression, with deregulated expression and established involvement in several human malignancies. Many circRNAs have been shown to bind microRNAs (miRNAs), thus making miRNAs unavailable to regulate protein-coding gene expression. Moreover, some of them have been described to interact with chromatin and affecting its structure. Despite their pivotal roles in the development and progression of cancer, only three studies have investigated their expression and role(s) in chronic lymphocytic leukemia (CLL), so far. The products of BCL2 and its homologues, including BAX and BCL2-like 12 (BCL2L12), are implicated in CLL, as apoptosis regulators. Moreover, expression levels of both BAX and BCL2L12 mRNAs are deregulated in CLL and are associated with patients’ response to therapy and overall survival. However, to the best of our knowledge, nothing is known about circRNAs produced by these two genes and their regulatory potential. Aims: We sought to further elucidate the contribution of both BAX and BCL2L12 in CLL by unraveling the identity of their circRNAs in a B-cell leukemic cell line (EHEB) and by studying in silico their potential interactions with miRNAs having an already established role in CLL. Methods: EHEB cells were propagated according to ATCC instructions. After total RNA extraction, 2μg of total RNA were reverse-transcribed using random hexamers. Next, nested PCR with divergent primers was performed, starting from each exon of BAX and BCL2L12 genes; thus, only cDNAs from BAX and BCL2L12 circRNAs were amplified, respectively. After having mixed and purified all nested-PCR products, nanopore sequencing libraries were built according to an optimized protocol. Following nanopore sequencing and basecalling in a MinION Mk1C 3rd-generation sequencer, sequencing reads were aligned and corrected using Minimap2 and TranscriptClean, respectively. circRNA sequences were identified using an in-house–built, PERL-based algorithm. A single-molecule resolution fluorescent in situ hybridization (FISH) method called circFISH was used to visualize the circRNA distribution in the cells. Lastly, miRDB was used to predict interactions of the most abundant BAX and BCL2L12 circRNAs with miRNAs. Results: We discovered 18 and 21 novel circRNAs produced by BAX and BCL2L12 genes, respectively, in this leukemic cell line. The exon structure of these novel circRNAs showed remarkable diversity, also comprising genomic regions that have previously been considered as intronic, as they are not included in messenger RNAs (mRNAs). Interestingly, one of the two most abundant BAX circRNAs is composed of a single exon, also spanning two introns. Similar intron retentions have been observed in a few BCL2L12 circRNAs. The gamut of miRNAs predicted to be bound by novel circRNAs includes miR-484, miR-181a-2-3p and miR-214-3p. Particularly, the inhibition of miR-214-3p is associated with elevated apoptosis in leukemic B-cells. Summary/Conclusion: Alternative splicing and back-splicing mechanisms of the primary BAX and BCL2L12 transcripts produce multiple distinct circRNAs in this B-cell CLL cell line. Interestingly, one of the two most abundant circular transcripts of BAX is a circRNA spanning three exons and two intervening introns of this gene. Lastly, several miRNAs are predicted to be sequestered by circRNAs of either of these apoptosis-related genes, including miR-181 family members that have been associated with chemosensitivity. Our data suggest a multifaceted role of BAX and BCL2L12 circRNAs in B-cell CLL.

Reconstruction of Full-Length circRNA Sequences Using Chimeric Alignment Information.

Circular RNAs (circRNAs) are RNA molecules formed by joining a downstream 3 splice donor site and an upstream 5 splice acceptor site. Several recent studies have identified circRNAs as potential biomarker for different diseases. A number of methods are available for the identification of circRNAs. The circRNA identification methods cannot provide full-length sequences. Reconstruction of the full-length sequences is crucial for the downstream analyses of circRNA research including differential expression analysis, circRNA-miRNA interaction analysis and other functional studies of the circRNAs. However, a limited number of methods are available in the literature for the reconstruction of full-length circRNA sequences. We developed a new method, circRNA-full, for full-length circRNA sequence reconstruction utilizing chimeric alignment information from the STAR aligner. To evaluate our method, we used full-length circRNA sequences produced by isocirc and ciri-long using long-reads RNA-seq data. Our method achieved better reconstruction rate, precision, sensitivity and F1 score than the existing full-length circRNA sequence reconstruction tool ciri-full for both human and mouse data.