Candidate circRNAs Research Articles

Aberrant dysregulated circular RNAs in the peripheral blood mononuclear cells of patients with rheumatoid arthritis revealed by RNA sequencing: novel diagnostic markers for RA

Circular RNAs (circRNAs) represent a newly identified class of non-coding RNAs that have been shown to be involved in several diseases, including autoimmune diseases. Two studies have revealed the aberrant circRNA expression profiles in the peripheral blood mononuclear cells (PBMCs) of patients with rheumatoid arthritis (RA) by microarrays. However, due to the intrinsic defects of microarrays, such as their inability to detect unidentified circRNAs, we examined the circRNA expression profiles in the PBMCs from four RA patients and three healthy controls by RNA sequencing (RNA-seq) and further explored the value of circRNAs in diagnosing RA. The results showed 71 markedly dysregulated circRNAs, including 41 upregulated and 30 downregulated circRNAs; these data included several previously unidentified candidate circRNAs. Gene Ontology and pathway annotation revealed that the most altered pathways and genes were associated with inflammation and transcriptional activity, such as the TNF pathway. The selected dysregulated circRNAs were verified by qRT-PCR in the PBMCs of 32 RA patients and 20 healthy controls, and the results indicated that hsa_circ_0000396 and hsa_circ_0130438 were downregulated in the RA group versus the healthy group, consistent with the RNA-seq data. The area under the receiver operating characteristic curve indicated the diagnostic value of both circRNAs for RA. Our results identified aberrant dysregulated circRNAs in RA patients, including several identified circRNAs, and the diagnostic value of circRNAs for RA, suggesting the superiority of RNA-seq versus microarrays for screening differentially expressed circRNAs and further strengthening the potential diagnostic value of circRNAs for the diagnosis of RA.

Hsa_circ_0000479 as a Novel Diagnostic Biomarker of Systemic Lupus Erythematosus.

Background: Accumulating evidence suggests that differentially expressed non-coding circular RNAs (circRNAs) play critical roles in the progress of autoimmune diseases. However, the role of circRNAs in systemic lupus erythematosus (SLE) remains unclear.Methods: We initially used next-generation sequencing (NGS) to comprehensively analyze circRNA expression profiles in peripheral blood mononuclear cells (PBMCs) from 10 SLE patients, stratified by their disease activity characteristics (stable or active SLE), and 10 healthy controls (HCs). Candidate circRNAs identified were first validated by quantitative reverse-transcription (qRT)-PCR in PBMC samples from a training-phase cohort of five SLE patients and five HCs. The significantly dysregulated circRNAs were then confirmed by qRT-PCR in a validation cohort of 23 SLE patients and 21 HCs, and in an external validation cohort with 64 SLE patients, 58 HCs, and 50 patients with rheumatoid arthritis (RA). In addition, we conducted bioinformatics analysis and western blotting investigating the relationships between the candidate circRNAs and SLE progression.Results: Multilayer integrative analysis of circRNA regulation showed that 84 circRNAs were upregulated and 30 were downregulated in patients with SLE compared with HCs. We then analyzed the intersection of these differentially expressed circRNAs in an SLE-stable cohort, an SLE-active cohort, and HCs. This enabled us to narrow down dysregulated circRNAs to 15 upregulated circRNAs. Only hsa_circ_0000479 was significantly upregulated in PBMCs of patients with SLE compared with HCs (P < 0.05). Furthermore, the diagnostic potential of hsa_circ_0000479 expression to distinguish SLE patients from HCs and RA patients was also significantly increased in the validation-phase and external-validation-phase cohorts (P < 0.05). When distinguishing SLE patients from HCs, the diagnostic specificities of hsa_circ_0000479 were 0.619 and 1.0 in two validation cohorts, respectively (AUCs = 0.731 and 0.730, respectively). It was also significantly increased in either stable SLE patients or active SLE patients compared with HCs in these two cohorts (P < 0.05). We also used bioinformatics analysis to show that hsa_circ_0000479 regulates SLE progression by modulating metabolic pathways and the Wnt signaling pathway. Western blotting revealed that the expression of Wnt-16 protein was significantly decreased in SLE.Conclusion: Our results suggest that hsa_circ_0000479 has potential as a novel biomarker for the diagnosis of SLE.

Comprehensive expression profiles and bioinformatics analysis reveal special circular RNA expression and potential predictability in the peripheral blood of humans with idiopathic membranous nephropathy.

The etiology of idiopathic membranous nephropathy (IMN) is considered to be closely associated with immunoregulation and genetic factors. Circular RNAs (circRNAs) have been found to regulate gene expression in various organisms, and to play an important role in multiple physiological and pathological processes, which may be involved in the pathogenesis of IMN. The purpose of the present study was to investigate the potential relationship between circRNAs in peripheral blood and disease. The diagnoses of IMN were confirmed using electron microscopy and immunofluorescence. Total RNA was isolated and microarray analysis was used to detect the expression levels of circRNAs in the peripheral blood of patients with IMN and in normal subjects. Selected genes from the microarray were selected and verified by reverse transcription-quantitative (RT-q)PCR. Bioinformatics tools were applied for further functional evaluation, and the potential disease predictability of circRNAs was determined using receiver-operating characteristic (ROC) curves. The results showed that a total of 955 differentially expressed circRNAs were found in blood samples, 645 of which were upregulated and 310 which were downregulated. In total, five candidate circRNAs were validated using RT-qPCR analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses identified numerous types of target genes and their corresponding microRNAs (miRNAs). The miRNAs identified were involved in biological processes and enriched in multiple important pathways, including the mitogen-activated protein kinase, transforming growth factor-β and Ras signaling pathways. The levels of circ_101319 were significantly higher (P<0.001) and exhibited promising diagnostic value in patients with IMN (area under ROC =0.89). The co-expression network constructed for circ_101319 indicated that it may be associated with membranous nephropathy-related pathways by mediating miRNAs. In conclusion, the present study revealed the expression and functional profile of differentially expressed circRNAs in the peripheral blood of patients with IMN, and provided new perspectives to predict and elucidate the development of IMN.

CircRNA expression profiles and function prediction in peripheral blood mononuclear cells of patients with acute ischemic stroke.

Most circular RNAs (circRNAs) belong to a novel class of noncoding RNAs that are produced by back-splicing reactions, and they regulate physiological and pathophysiological processes in human disease. Although circRNA expression has been shown to be altered in the ischemic cerebral tissue in animal studies, the expression profile of circRNA in the patients with acute ischemic stroke (AIS) has not been investigated to date. In this investigation, high-throughput sequencing was carried out to compare the circRNA expression of peripheral blood mononuclear cells (PBMCs) from five patients with AIS and five healthy subjects. A total of 521 circRNAs were expressed differentially between the patients with AIS and healthy controls (p < .05, fold difference ≥2) including 373 upregulated circRNAs and 148 downregulated circRNAs in patients with AIS compared to controls. Thirteen candidate circRNAs were verified by quantitative real-time polymerase chain reaction (qRT-PCR). Bioinformatics analyses showed that these differentially expressed circRNAs were highly conserved, as well as eight circRNAs that were confirmed by qRT-PCR containing binding sites to multiple microRNAs. Kyoto Encyclopedia of Genes and Genomes pathway enrichment and gene ontology analyses indicated that the aberrantly expressed circRNAs participated in many pathophysiological processes of AIS, especially regarding inflammation and immunity. In conclusion, patients with AIS differentially express certain circRNAs in PBMCs, which may be diagnostic biomarkers or potential therapeutic targets.

Long and Repeat-Rich Intronic Sequences Favor Circular RNA Formation under Conditions of Reduced Spliceosome Activity.

SummaryCircular RNAs (circRNAs), an important class of regulatory RNAs, have been shown to be the most prevalent in the brain compared with other tissues. However the processes governing their biogenesis in neurons are still elusive. Moreover, little is known about whether and how different biogenesis factors work in synchrony to generate neuronal circRNAs. To address this question, we pharmacologically inhibited the spliceosome and profiled rat neuronal circRNAs using RNA sequencing. We identified over 100 circRNAs that were up-regulated and a few circRNAs that were down-regulated upon spliceosome inhibition. Bioinformatic analysis revealed that up-regulated circRNAs possess significantly longer flanking introns compared with the un-changed circRNA population. Moreover, the flanking introns of up-regulated circRNAs harbor a higher number of distinct repeat sequences and more reverse complementary motifs compared with the unchanged circRNAs. Taken together, our data demonstrate that the biogenesis of circRNAs containing distinct intronic features becomes favored under conditions of limited spliceosome activity.

Circulating plasma circular RNAs as novel diagnostic biomarkers for congenital heart disease in children.

ObjectiveThe diagnostic value of circulating circular RNAs (circRNAs) has received more and more attention. However, little has been reported about their potential in the diagnosis of congenital heart diseases (CHD). In this study, we explored differential expression of circRNAs from children with CHD to evaluate their potential as clinical biomarkers.MethodsWe established a discovery cohort (four CHD cases; four matched healthy controls) and a validation cohort (40 CHD cases; 40 matched healthy controls). Microarray expression analysis was performed on the discovery set to identify candidate circRNAs. Candidates were further validated in the validation set. The diagnostic accuracy of circRNAs was determined by receiver operating characteristic (ROC) analysis. Gene ontology (GO), pathway, and network analysis were performed to predict a network of circRNA/miRNA and target mRNAs related to CHD.ResultsThe top seven significantly differentially expressed CHD‐associated circRNAs were validated by RT‐PCR as follows: hsa_circRNA_004183, hsa_circRNA_079265, hsa_circRNA_105039, hsa_circRNA_404686, hsa_circRNA_101050, hsa_circRNA_100787, and hsa_circRNA_101328. Three significantly down‐regulated circRNAs (hsa_circRNA_004183, hsa_circRNA_079265, and hsa_circRNA_105039) were identified with area under curve (AUC) values of 0.758, 0.809, and 0.907, respectively; the combination had an AUC of 0.965. An interaction network was constructed by 43 circRNAs, 9 miRNAs, and 29 mRNAs, which involved in heart development.ConclusionsWe identified three circRNAs under‐expressed in plasma from children with CHD. These circRNAs may be crucial in the development of CHD and may serve as novel non‐invasive biomarkers for the diagnosis of CHD in children.

Hsa_circ_0058124 promotes papillary thyroid cancer tumorigenesis and invasiveness through the NOTCH3/GATAD2A axis

BackgroundDespite a good and overall prognosis, papillary thyroid cancer (PTC) can still affect the quality of life of many patients, and can even be life-threatening due to its invasiveness and metastasis. Emerging studies demonstrate that circular RNAs (circRNAs) participate in the regulation of various cancers. However, the circRNA profile in invasive PTC is still not well understood.MethodsCompeting endogenous RNA (ceRNA) microarrays were performed to determine circRNAs contributed to the tumorigenesis and invasiveness of PTC. Bioinformatics methods were used to narrow down the candidate circRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) assays revealed a significant upregulation of hsa_circ_0058124 in PTC tissue and a close correlation with a poor prognosis for PTC patients. RNA fluorescence in situ hybridization and Cell fractionation assay were used to investigate the subcellular location of hsa_circ_0058124. Then, we examined the functions of hsa_circ_0058124 in PTC by cell proliferation, cell cycle, apoptosis, migration and invasion assay. Mechanistically, RNA sequencing and GSEA analysis were applied to predict the downstream pathway of hsa_circ_0058124. Dual-luciferase report assays were used to explore the potential miRNA sponge role of hsa_circ_0058124. Western blotting, cell proliferation, cell cycle, cell apoptosis, migration and invasion, and mouse xenograft assay were used to validate the effects of hsa_circ_0058124/NOTCH3/GATAD2A axis on PTC progression.ResultsIn the current study, a novel hsa_circ_0058124 on 2q35 was identified and explored in PTC. Hsa_circ_0058124 is associated with the malignant features and poor outcomes of PTC patients. Hsa_circ_0058124 acts as an oncogenic driver that promotes PTC cell proliferation, tumorigenicity, tumor invasion, and metastasis, which functions as a competing endogenous RNA to modulate miRNA-218-5p and its target gene NUMB expression, and consequently with repression of the NOTCH3/GATAD2A signaling axis in vitro and in vivo.ConclusionsThis study unveils a novel biomarker panel consisting of the hsa_circ_0058124/NOTCH3/GATAD2A axis which is critical for PTC tumorigenesis and invasiveness and may represent a novel therapeutic target for intervening in PTC progression.

Abstract 1820: Circular RNAs contribute to neuroblastoma pathogenesis

Abstract Circular RNAs (circRNAs), a noncoding RNA class originating from alternative splicing, are highly abundant in neural tissues and can regulate gene expression by binding to and inhibiting microRNAs and RNA-binding proteins. We explored whether circRNAs influence pathogenesis in neuroblastoma, the most common solid extracranial tumor of childhood. We performed whole-transcriptome sequencing of primary neuroblastoma samples to identify candidate circRNAs, which were validated in a neuroblastoma cell line panel. Overexpression and knockdown models were created to investigate their impact on cell viability, proliferation, apoptosis and differentiation. We identified 4,482 unique circRNA backsplicing junctions involving 2,080 genes in 69 neuroblastoma samples from all risk groups. Candidate circRNA expression did not correlate with host gene expression, indicating independent regulatory mechanisms. Principal component analysis was conducted using tumor circRNA expression. MYCN-amplified tumors clustered tightly together, demonstrating that variations in circRNA expression identify MYCN-amplified neuroblastomas as do gene expression patterns. Comparing our RNA sequencing data with other cancers and healthy fetal brain tissue revealed a circRNA subset specifically upregulated in neuroblastoma that included a circRNA derived from the ARID1A tumor suppressor gene. Cell fractionization and RNA FISH localized circARID1A to the cytoplasm, where it could interact with miRNAs and RNA-binding proteins for functional consequences. Specific circARID1A knockdown reduced cell numbers and viability, and induced neurite outgrowth and differentiation markers. Neither knockdown, nor overexpression of circARID1A influenced ARID1A mRNA and protein levels. Ongoing efforts applying bioinformatics methods to investigate the mechanism of action have identified several binding sites of microRNAs and RNA-binding proteins enriched in the circARID1A sequence. We demonstrate that circRNAs are expressed in neuroblastomas unrelated to host gene expression, and that their expression can be used to cluster MYCN-amplified tumors. As one in a set of upregulated circRNAs, circARID1A acts in a tumor-promoting manner in neuroblastoma cell lines independent of the host gene. Citation Format: Filippos Klironomos, Clara Danssmann, Julian Naderi, Annika Winkler, Uschi Luz, Patrick Hundsdoerfer, Angelika Eggert, Joern Toedling, Falk Hertwig, Johannes H. Schulte, Steffen Fuchs. Circular RNAs contribute to neuroblastoma pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1820.

Abstract LB-307: Autophagy-associated circRNA AUACA1 mediates the proliferation of breast cancer cells via a miR-1275/ATG7/autophagic flux axis

Abstract Introduction: Autophagy plays a critical role in cancer initiation and progression. CircRNAs were recently identified as new molecules in regulation of cancer cell biology. However, it remains unclear whether circRNAs are involved in the process of autophagy and regulate cancer biology via autophagy. Methods: We performed circRNAs microarray to screen autophagy-associated circRNAs in hypoxia-induced autophagy model and qRT-PCR was used to verify the expression level of candidate circRNAs after autophagy induction in both hypoxia and starvation induced autophagy models. RNase R digestion assay and Poly A detective experiment were used to verify the circular characteristic of circRNA candidates. The proliferative effects of knocking down and overexpressing circRNA in breast cancer cells were tested by colony formation and cell counting assays. Western Blot was used to test the protein level of LC3 and ATG7 in cancer cells. Ago2 RIP assay was used to test the miRNA sponge potential of circRNA candidates. CHiRP assays, miRNA pull down assays and dual-luciferase reporter system assays were used to verify the miRNA sponge of circRNA candidate. The location and expression levels of AUACA1 and ATG7 in human breast tissue was detected by ISH and IHC respectively. Survival rates and curves in a breast cancer cohort of 109 patients were determined by the Kaplan-Meier method and the comparison of survival differences was evaluated by using the log-rank test. Results: In our study, we found that breast cancer cells after autophagy induction displayed specific circRNA profiles. Among the altered circRNAs, upregulation of AUACA1 (named Autophagy Associated CircRNA 1) was most obvious in both hypoxia and starvation-induced autophagy. Moreover, AUACA1 upregulation was parallel to the degree of autophagy in breast cancer cells, while inhibition of autophagy did not affect the level of the AUACA1. Rnase R digestion and Poly A detective experiment showed that character of AUACA1 was circular rather than linear. Nuclear plasma separation assay and in-situ hybridization showed that AUACA1 mostly located in the cytoplasm. Knocking down of AUACA1 significantly inhibited the proliferation of breast cancer cells, but did not affect cell apoptosis and migration. Knocking down AUACA1 decreased autophagic level by detecting LC3-II. Proliferative assay demonstrated that AUACA1 promoted proliferation via autophagy in MDA-MB-231. Ago2 RIP assay significantly enriched AUACA1, indicating AUACA1 may worked as miRNA sponge. CHiRP and miRNAs microarray proved that AUACA1 worked as miR-1275 sponge. MiRNA pull down assay and dual-luciferase reporter system assays also verified that AUACA1 worked as miR-1275 sponge. MiR-1275 affected ATG7 expression at both RNA and protein level after transfecting miR-1275 mimics in MDA-MB-231. In addition, clinical data indicated that AUACA1 expression level was positively correlated with ATG7 and Ki-67 index. High expression of AUACA1 was correlated with high tumour burden and poor disease-free survival in 109 breast cancer patients. Conclusion: Our findings indicate that AUACA1 is an autophagy–associated circRNA and plays a critical role in promoting the proliferation of breast cancer cell via miR- 1275/ATG7/autophagic axis. AUACA1 may be a potential prognostic biomarker for breast cancer. Citation Format: Gehao Liang, Yun Ling, Zihao Liu, Maryam Mehrpour, Ahmed Hamai, Zhenluan Tian, Luyuan Tan, Wenjing Zhong, Wanyi Lin, Patrice Codogno, Erwei Song, Chang Gong. Autophagy-associated circRNA AUACA1 mediates the proliferation of breast cancer cells via a miR-1275/ATG7/autophagic flux axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-307.

Circular RNA circSLC8A1 acts as a sponge of miR-130b/miR-494 in suppressing bladder cancer progression via regulating PTEN

BackgroundCircular RNAs (circRNAs) are a novel class of endogenous noncoding RNAs formed by a covalently closed loop, and increasing evidence has revealed that circRNAs play crucial functions in regulating gene expression. CircSLC8A1 is a circRNA generated from the SLC8A1 gene. Currently, the role and underlying molecular mechanisms of circSLC8A1 in bladder cancer remain unknown.MethodsThe differentially expressed circRNAs were identified from RNA-sequencing data, and circSLC8A1 was determined as a new candidate circRNA. qRT-PCR was used to detect the expression of circRNAs, miRNAs and mRNAs in human tissues and cells. RNA pull-down assay and luciferase reporter assay were used to investigate the interactions between the specific circRNA, miRNA and mRNA. The effects of circSLC8A1 on bladder cancer cells were explored by transfecting with plasmids in vitro and in vivo. The expression of PTEN was detected by Western blot. The biological roles were measured by wound healing assay, transwell assay, and CCK-8 assay.ResultsIn the present study, we found that circSLC8A1 was down-regulated in bladder cancer tissues and cell lines, and circSLC8A1 expression was associated with the pathological stage and histological grade of bladder cancer. Over-expression of circSLC8A1 inhibited cell migration, invasion and proliferation both in vitro and in vivo. Mechanistically, circSLC8A1 could directly interact with miR-130b/miR-494, and subsequently act as a miRNA sponge to regulate the expression of the miR-130b/miR-494 target gene PTEN and downstream signaling pathway, which suppressed the progression of bladder cancer.ConclusionsCircSLC8A1 acts as a tumor suppressor by a novel circSLC8A1/miR-130b, miR-494/PTEN axis, which may provide a potential biomarker and therapeutic target for the management of bladder cancer.

Comprehensive Analysis of lncRNAs and circRNAs Reveals the Metabolic Specialization in Oxidative and Glycolytic Skeletal Muscles

The biochemical and functional differences between oxidative and glycolytic muscles could affect human muscle health and animal meat quality. However, present understanding of the epigenetic regulation with respect to lncRNAs and circRNAs is rudimentary. Here, porcine oxidative and glycolytic skeletal muscles, which were at the growth curve inflection point, were sampled to survey variant global expression of lncRNAs and circRNAs using RNA-seq. A total of 4046 lncRNAs were identified, including 911 differentially expressed lncRNAs (p < 0.05). The cis-regulatory analysis identified target genes that were enriched for specific GO terms and pathways (p < 0.05), including the oxidation-reduction process, glycolytic process, and fatty acid metabolic. All these were closely related to different phenotypes between oxidative and glycolytic muscles. Additionally, 810 circRNAs were identified, of which 137 were differentially expressed (p < 0.05). Interestingly, some circRNA-miRNA-mRNA networks were found, which were closely linked to muscle fiber-type switching and mitochondria biogenesis in muscles. Furthermore, 44.69%, 39.19%, and 54.01% of differentially expressed mRNAs, lncRNAs, and circRNAs respectively were significantly enriched in pig quantitative trait loci (QTL) regions for growth and meat quality traits. This study reveals a mass of candidate lncRNAs and circRNAs involved in muscle physiological functions, which may improve understanding of muscle metabolism and development from an epigenetic perspective.

Prediction of circRNAs Based on the DNA Methylation-Mediated Feature Sponge Function in Breast Cancer

Background: Several studies have found that DNA methylation is associated with transcriptional regulation and affect sponge regulation of non-coding RNAs in cancer. The integration of circRNA, miRNA, DNA methylation and gene expression data to identify sponge circRNAs is important for revealing the role of DNA methylation-mediated regulation of sponge circRNAs in cancer progression. Method: We established a DNA methylation-mediated circRNA crosstalk network by integrating gene expression, DNA methylation and non-coding RNA data of breast cancer in TCGA. Four modules (26 candidate circRNAs) were mined. Next, 10 DNA methylation-mediated sponge circRNAs (sp_circRNAs) and 5 sponge driver genes (sp_driver genes) in breast cancer were identified in the CMD network using a computational process. Findings: Among the identified genes, ERBB2 was associated with six sponge circRNAs, which illustrates its better sponge regulatory function. Survival analysis showed that DNA methylations of 10 sponge circRNA host genes are potential prognostic biomarkers in the TCGA dataset (p=0.0239) and GSE78754 dataset (p=0.0377). In addition, the DNA methylation of two sponge circRNA host genes showed a significant negative correlation with their driver gene expressions. Interpretation: We developed a strategy to predict sponge circRNAs by DNA methylation mediated with playing the role of regulating breast cancer sponge driver genes. Funding Statement: This work was supported by National Natural Science Foundation of China [grant number 81573021], the Natural Science Foundation of Heilongjiang Province [grant number ZD2015003], and the Heilongjiang Science and Technology Plan Project [YS17C22]. Declaration of Interests: We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and company that could be construed as influencing the position presented in, or the review of, this manuscript. Ethics Approval Statement: The authors declare: Not applicable.

CircRNA hsa_circ_0004585 as a potential biomarker for colorectal cancer.

Objective: Circular RNAs (circRNAs) are involved in regulating of carcinogenesis of various cancer cells. However, the function of circRNAs in colorectal cancer (CRC) has remained largely unknown. This study investigated the characteristic expression of circRNAs in CRC and adjacent normal tissues, analyzed the miRNAs related to candidate circRNAs, and studied the correlation between circRNAs with clinical data of CRC.Methods: Human CircRNA microarray has been applied to screen the expressions of circRNAs of the CRC tissues and adjacent normal tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) verified the candidate circRNAs in CRC tissue and patients’ peripheral blood. The circRNA array data were analyzed by GeneSpring 13.0 (Agilent) software. The diseases, pathways and functional enrichment analysis of these genes were performed using the KEGG system. In addition, the circRNA-miRNA network was constructed based on the miRanda-3.3 software. Statistical analysis was performed with SPSS23.0, GraphPad Prism, and Sigmaplot software.Results: In total, 13,198 circRNAs were identified as distinct between CRC and adjacent normal tissues, including 6,697 upregulated and 6,501 downregulated genes. Based on scores, six of them were selected for further verification in CRC tissues and peripheral blood. The hsa_circ_0004585 expression was significantly upregulated both in CRC patients tissue and peripheral blood. Hsa_circ_0004585 was positively correlated with patient’s tumor size, indicating the function of hsa_circ_0004585 in CRC carcinogenesis and metastasis.Conclusion: Hsa_circ_0004585 could be a potential biomarker for diagnosis of CRC.

Circular RNA circ KMT2E is up-regulated in diabetic cataract lenses and is associated with miR-204-5p sponge function

Covalently closed, single-stranded circular RNAs (circRNAs) are a class of newly discovered endogenous RNAs involved in the pathological process of various types of diseases through sponging microRNAs (miRNAs). However, the role of circRNAs in diabetic cataract (DC) is unclear. Our previous studies have demonstrated that miR-204-5p was expressed more lowly in DC than the normal controls, but whether it relates to the sponge function of circRNAs remains unknown. This study aimed to identify differentially expressed circRNAs in DC tissues and investigate the interaction between circRNAs and miR-204-5p in the development of cataract. RNA-sequencing based circRNA expression profiling was determined in DC lens tissues as well as transparent lens tissues, and 1063 circRNAs were significantly differentially expressed in the DC group compared to the normal control group (p ≤ 0.05, fold change ≥ 2.0). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were applied to predict the function of these differentially expressed circRNAs, and the top ten enriched GO entries and KEGG pathways were annotated. Expression levels of the two candidate circRNAs having conserved interaction with miR-204-5p were validated by qRT-PCR, showing that the change direction of these circRNAs was consistent with the RNA-sequencing data. Moreover, circKMT2E was up-regulated more than two folds in DC lens tissues compared with normal tissues, exhibiting an expression trend opposite to miR-204-5p. Bio-informatics analysis showed that there were totally four seed sequences of circKMT2E on hsa-miR-204-5p. Thus, we speculated that circKMT2E may function as a sponge molecule of miR-204-5p and play a role in the pathogenesis of DC. Although the exact mechanisms need further validation, our study found that the differentially expressed circRNAs was involved in the pathogenesis of DC, which can provide a new target for non-surgical treatment of DC.

A novel strategy of identifying circRNA biomarkers in cardiovascular disease by meta‐analysis

Circular RNAs (circRNAs) are stable and abundantly expressed in vivo but are abnormally expressed in several diseases. This study aimed to identify circRNAs acting as potential biomarkers for cardiovascular disease (CVD). Research were retrieved from the articles published by September 2018 in eight databases to compare circRNA expression profiles between CVD and non-CVD in human and animal models. Meta-analysis under a random effects model was conducted. Subgroup analysis of tissue, species, and disease-specific circRNAs was examined. Sensitivity analysis was performed to explain the uncertainty among all studies. Diagnostic accuracy of circRNAs in CVD was analyzed to testify the discriminative ability. Bioinformatics analysis including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was conducted. Among 6,284 differentially expressed circRNAs from 32 original studies, only 322 circRNAs were reported in three or more studies. The meta-analysis identified 63 significantly dysregulated circRNAs, 44 upregulated and 19 downregulated. Among the tissue-specific or disease-specific circRNAs identified in the subgroup analysis, two circRNAs (circCDKN2BAS and circMACF1) showed the potential to be circulating biomarkers for CVD. Sensitivity analysis demonstrated 69% of circRNAs were in conformity with the overall analysis. The pooled diagnostic odds ratio was 2.94 (95% confidence interval [CI], 2.35-3.58), and the overall area under the curve value was 0.86 (95% CI, 0.83-0.89). GO and KEGG enrichment analyses indicated that the target genes of circRNAs participate in cardiogenesis-related processes and pathways. This study demonstrates circRNAs have a high diagnostic value as potential biomarkers for CVD, and two candidate circRNAs, circCDKN2BAS and circMACF1, are potential circulating biomarkers for CVD diagnosis and treatment.

Microarray analysis reveals the changes of circular RNA expression and molecular mechanism in acute lung injury mouse model.

Acute lung injury (ALI) is a severe disease with sudden onset, rapid progression, poor treatment response, and high mortality. An increasing number of studies had found that circular RNAs (circRNAs) has significant functions in various diseases, while the role of circRNAs in ALI is not yet clear. The purpose of this study was to find circRNAs related to ALI and their mechanism of action. Expression profiles of lung circRNAs and messenger RNAs (mRNAs) were analyzed by microarray in the ALI mice models and healthy controlled mice. Differentially expressed RNAs were identified, function and pathways were analyzed by bioinformatics analysis. Moreover, the results of the microarray were verified by real-time PCR. We identified 2262 differentially expressed mRNAs and 581 circRNAs between ALI mice and control. Validation of candidate circRNAs by real-time PCR indicates that the majority of circRNAs identified by microarray are reliable and worthy of further study. ALI induced circRNAs primarily function in the metabolic regulatory process. Moreover, differentially expressed circRNAs were mainly involved in signaling pathways of mitogen-activated protein kinases, focal adhesion, FoxO, neurotrophin, and Wnt. In addition, a competitive endogenous RNA network was constructed to further interpret the molecular mechanism of ALI. This study observed significantly changed circRNAs profiles in LPS-induced mouse model and revealed a potential role of circRNAs in ALI.

Circular RNA rno_circ_0004002 regulates cell proliferation, apoptosis, and epithelial-mesenchymal transition through targeting miR-342-5p and Wnt3a in anorectal malformations.

Circular RNAs (circRNAs) are a novel class of noncoding RNAs that have regulatory functions in varieties of diseases. The purpose of this study was to investigate the potential role of circRNAs in anorectal malformations (ARM). With sequencing analysis, 78 circRNAs were identified to be differentially expressed on gestational day (GD) 19 in ARM and normal anorectal tissues, including 41 upregulated and 37 downregulated circRNAs. Among the downregulated circRNAs, rno_circ_0004002 was selected as the candidate circRNA. Functionally, silencing of rno_circ_0004002 was correlated with suppression of proliferation, and promotion of apoptosis and epithelial-mesenchymal transition (EMT) in anorectal development. For the mechanism investigation, bioinformatic prediction and luciferase reporter assay revealed that rno_circ_0004002 was proved to be a sponge of miR-342-5p, and miR-342-5p could target 3'-UTR of Wnt3a to negatively regulate its expression. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) results showed that the expression of miR-342-5p was significantly increased in ARM anorectal tissues compared with normal tissues on GD19 and GD21, and the expression of Wnt3a demonstrated the opposite on GD21. In addition, rescue assays disclosed that miR-342-5p inhibitor could reverse the role of rno_circ_0004002 knockdown on the repression of anorectal development. In summary, our study shed light on the regulatory mechanism of rno_circ_0004002 in cell proliferation, apoptosis, and EMT via sponging miR-342-5p, which downregulated the Wnt3a expression in anorectal development. We also suggested that GD19 and GD21 was the crucial time in the progression of ARM, and rno_circ_0004002/miR-342-5p/Wnt3a might serve as potential therapeutic targets for ARM.

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

Background and aimsThe 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. MethodsThe 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. ResultsA 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. ConclusionsWe 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 SNX29 Sponges miR-744 to Regulate Proliferation and Differentiation of Myoblasts by Activating the Wnt5a/Ca2+ Signaling Pathway

Myogenesis is a complex and precisely orchestrated process that is highly regulated by several non-coding RNAs and signal pathways. Circular RNAs (circRNAs) represent a novel subclass of endogenous non-coding RNAs that have been identified in multiple species and tissues and play a vital role in post-transcriptional regulation in eukaryotes, but the precise molecular mechanism of action remains largely unknown. Here, we screened a candidate circRNA derived from the SNX29 gene, termed circSNX29 from our previous circRNAs sequencing data of bovine skeletal muscle, and further characterized its regulation and function during muscle development. The overexpression of circSNX29 facilitated myoblasts differentiation and inhibited cell proliferation. Computational analysis using RNAhybrid showed the potential for circSNX29 to sponge to miR-744 with nine potential binding sites. We tested this via a luciferase screening assay and found that circSNX29 directly interacted with miR-744 and downregulation of miR-744 efficiently reversed the suppression of Wnt5a and CaMKIIδ. Importantly, through the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis, Fluo-4, AM, cell permeant-calcium ion fluorescent probing, and western blotting assays, we found that overexpression of Wnt5a and circSNX29 activated the non-canonical Wnt5a/Ca2+ pathway. Overall, the evidence generated by our study elucidates the regulatory mechanisms of circSNX29 to function as a sponge for miRNA-744 in bovine primary myoblasts.

Identification of Key Genes and Circular RNAs in Human Gastric Cancer.

BackgroundGlobally, gastric cancer (GC) is the third most common source of cancer-associated mortality. The aim of this study was to identify key genes and circular RNAs (circRNAs) in GC diagnosis, prognosis, and therapy and to further explore the potential molecular mechanisms of GC.Material/MethodsDifferentially expressed genes (DEGs) and circRNAs (DE circRNAs) between GC tissues and adjacent non-tumor tissues were identified from 3 mRNA and 3 circRNA expression profiles. Functional analyses were performed, and protein–protein interaction (PPI) networks were constructed. The significant modules and key genes in the PPI networks were identified. Kaplan-Meier analysis was performed to evaluate the prognostic value of these key genes. Potential miRNA-binding sites of the DE circRNAs and target genes of these miRNAs were predicted and used to construct DE circRNA–miRNA–mRNA networks.ResultsA total of 196 upregulated and 311 downregulated genes were identified in GC. The results of functional analysis showed that these DEGs were significantly enriched in a variety of functions and pathways, including extracellular matrix-related pathways. Ten hub genes (COL1A1, COL3A1, COL1A2, COL5A2, FN1, THBS1, COL5A1, SPARC, COL18A1, and COL11A1) were identified via PPI network analysis. Kaplan-Meier analysis revealed that 7 of these were associated with a poor overall survival in GC patients. Furthermore, we identified 2 DE circRNAs, hsa_circ_0000332 and hsa_circ_0021087. To reveal the potential molecular mechanisms of circRNAs in GC, DE circRNA–microRNA–mRNA networks were constructed.ConclusionsKey candidate genes and circRNAs were identified, and novel PPI and circRNA–microRNA–mRNA networks in GC were constructed. These may provide useful information for the exploration of potential biomarkers and targets for the diagnosis, prognosis, and therapy of GC.