Key circRNAs Research Articles

Transcriptome Sequencing Reveal That Rno-Rsf1_0012 Participates in Levodopa-Induced Dyskinesia in Parkinson's Disease Rats via Binding to Rno-mir-298-5p.

Levodopa-induced dyskinesia (LID) is a common complication of chronic dopamine replacement therapy in the treatment of Parkinson’s disease (PD), and a noble cause of disability in advanced PD patients. Circular RNA (circRNA) is a novel type of non-coding RNA with a covalently closed-loop structure, which can regulate gene expression and participate in many biological processes. However, the biological roles of circRNAs in LID are not completely known. In the present study, we established typical LID rat models by unilateral lesions of the medial forebrain bundle and repeated levodopa therapy. High-throughput next-generation sequencing was used to screen circRNAs differentially expressed in the brain of LID and non-LID (NLID) rats, and key circRNAs were selected according to bioinformatics analyses. Regarding fold change ≥2 and p < 0.05 as the cutoff value, there were a total of 99 differential circRNAs, including 39 up-regulated and 60 down-regulated circRNAs between the NLID and LID groups. The expression of rno-Rsf1_0012 was significantly increased in the striatum of LID rats and competitively bound rno-mir-298-5p. The high expression of target genes PCP and TBP in LID rats also supports the conclusion that rno-Rsf1_0012 may be related to the occurrence of LID.

CircularRNA expression profiles of ovarian granulosa cells in advanced-age women explain new mechanisms of ovarian aging.

Aim: We aimed to determine the role of granulosa cells (GCs) circular RNA (circRNA) in ovarian aging. Methods: Ninewomen were recruited, including threediminished ovarian reserve young women, threeadvanced-aged women and threenormal ovarian reserve young women. The circRNA expression profiles of GCs were characterized by CLEAR software. Key circRNA were validated by quantitative reverse transcriptionPCR. Results: GCs in advanced-age group females exhibited active MHC class II-related biological processes. A total of 3575 circRNAs were found in the advanced age group. Hsa-circ-0031584 appears to be one of the important molecules regulating the mitotic process of GCs. Conclusion: The expression profiles of circRNAs exhibited obvious stage specificity with age which might contribute to ovarian aging progression.

Genome-Wide Expression Profiling and Networking Reveals an Imperative Role of IMF-Associated Novel CircRNAs as ceRNA in Pigs.

Intramuscular fat (IMF) deposition is a biological process that has a strong impact on the nutritional and sensorial properties of meat, with relevant consequences on human health. Pork loins determine the effects of marbling on the sensory attributes and meat quality properties, which differ among various pig breeds. This study explores the crosstalk of non-coding RNAs with mRNAs and analyzes the potential pathogenic role of IMF-associated competing endogenous RNA (ceRNA) in IMF tissues, which offer a framework for the functional validation of key/potential genes. A high-throughput whole-genome transcriptome analysis of IMF tissues from longissimus dorsi muscles of Large White (D_JN) and Laiwu (L_JN) pigs resulted in the identification of 283 differentially expressed circRNAs (DECs), including two key circRNAs (circRNA-23437, circRNA-08840) with potential binding sites for multiple miRNAs regulating the whole network. The potential ceRNA mechanism identified the DEC target miRNAs-mRNAs involved in lipid metabolism, fat deposition, meat quality, and metabolic syndrome via the circRNA-miRNA-mRNA network, concluding that ssc-mir-370 is the most important target miRNA shared by both key circRNAs. TGM2, SLC5A6, ECI1, FASN, PER1, SLC25A34, SOD1, and COL5A3 were identified as hub genes through an intensive protein-protein interaction (PPI) network analysis of target genes acquired from the ceRNA regulatory network. Functional enrichments, pathway examinations, and qRT-PCR analyses infer their implications in fat/cholesterol metabolism, insulin secretion, and fatty acid biosynthesis. Here, circRNAs and miRNA sequencing accompanied by computational techniques were performed to analyze their expressions in IMF tissues from the longissimus dorsi muscles of two pig breeds. Their target gene evolutionary trajectories, expression profiling, functional enrichments, subcellular localizations, and structural advances with high-throughput protein modeling, following genomic organizations, will provide new insights into the underlying molecular mechanisms of adipocyte differentiation and IMF deposition and a much-needed qualitative framework for future research to improve meat quality and its role as a biomarker to treat lipid metabolic syndromes.

CircRERE regulates the expression of GBX2 through miR-1299 and ZC3H13/N6-methyladenosine (m6A) to promote growth and invasion of hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is one of the most common malignant tumours in the world. Current studies have shown that circular RNAs (circRNAs) and N6-methyladenosine (m6A) methylation play important roles in the progression of HCC, but further studies are needed to confirm the underlying mechanisms. The expression of circRERE was significantly upregulated in HCC cells, and its downregulation reduced HCC cell viability and invasion while increasing apoptosis. Further study showed that circRERE bound directly to miR- 1299. After downregulating the expression of circRERE, miR-1299 expression was significantly enhanced, while the expression of its downstream target gene GBX2 was suppressed, indicating that circRERE promoted the expression of GBX2 through miR-1299. In addition, downregulation of circRERE expression significantly increased the m6A level of GBX2 and promoted the expression of methyltransferase ZC3H13, while overexpression of ZC3H13 significantly inhibited the expression of GBX2 but increased its m6A methylation. circRERE could regulate the m6A modification of GBX2 through ZC3H13, thus promoting the expression of GBX2.GBX2 was upregulated in HCC tissues, while miR-1299 and ZC3H13 were downregulated. MiR-1299 mimics, ZC3H13 overexpression or GBX2 siRNA significantly inhibited HCC cell viability, promoted apoptosis and reduced invasion; GBX2 exerted the opposite effects and could reverse the regulatory effects of miR-1299 or ZC3H13 on HCC cells. Therefore, circRERE promotes the growth and invasion of HCC cells by regulating the expression of GBX2 through miR-1299 and ZC3H13/m6A, indicating that it is a key circRNA in the progression of HCC.

Identification of circular RNAs in cardiac hypertrophy and cardiac fibrosis.

Cardiac hypertrophy initially serves as an adaptive response to physiological and pathological stimuli. Sustained hypertrophy progress to pathological cardiac hypertrophy, cardiac fibrosis and ultimately lead to heart failure, one of the leading medical causes of mortality worldwide. Intervention of pathological cardiac hypertrophy can effectively reduce the occurrence of heart failure. Abundant factors, such as adrenergic, angiotensin, and endothelin (ET-1) receptors, have been shown to participate in the regulation of pathological cardiac hypertrophy. Recently, an increasing number of studies have indicated that circRNA and circRNA-miRNA–mRNA network regulation is indispensable for the posttranscriptional regulation of mRNA in cardiac hypertrophy. In our study, the morphological, cardiac function and pathological changes during cardiac hypertrophy were investigated. RNA sequencing identified 93 circRNAs that were differentially expressed in the TAC_2w group, and 55 circRNAs in the TAC_4w group compared with the sham group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses identified several significant pathways, including hypertrophic cardiomyopathy, extracellular matrix (ECM)-receptor interaction and focal adhesion. Coexpression analyses were performed for differentially expressed circRNAs and differentially expressed mRNAs. Based on gene set enrichment analysis (GSEA), 8 circRNAs (mmu-Nfkb1_0001, mmu-Smad4_0007, mmu-Hecw2_0009, mmu-Itgbl1_0002, mmu-Lrrc2_0005, mmu-Cpeb3_0007, mmu-Ryr2_0040, and mmu-Rtn4_0001) involved in cardiac hypertrophy and cardiac fibrosis were identified. We validated some key circRNAs by qPCR. The crucial coexpression of circRNA–mRNA and its interaction with miRNA showed the possible mechanism of circRNAs in the process of cardiac dysfunction. Our results may provide promising targets for the treatment of pathological cardiac hypertrophy and fibrosis.

Integrative analysis of circRNA, miRNA, and mRNA profiles to reveal ceRNA regulation in chicken muscle development from the embryonic to post-hatching periods

BackgroundThe growth and development of skeletal muscle are regulated by protein-coding genes and non-coding RNA. Circular RNA (circRNA) is a type of non-coding RNA involved in a variety of biological processes, especially in post-transcriptional regulation. To better understand the regulatory mechanism of circRNAs during the development of muscle in chicken, we performed RNA-seq with linear RNA depletion for chicken breast muscle in 12 (E 12) and17 (E 17) day embryos, and 1 (D 1), 14 (D 14), 56 (D 56), and 98 (D 98) days post-hatch.ResultsWe identified 5755 differentially expressed (DE)-circRNAs during muscle development. We profiled the expression of DE-circRNAs and mRNAs (identified in our previous study) at up to six time points during chicken muscle development and uncovered a significant profile (profile 16) for circRNA upregulation during aging in muscle tissues. To investigate competing endogenous RNA (ceRNA) regulation in muscle and identify muscle-related circRNAs, we constructed a circRNA-miRNA-mRNA regulatory network using the circRNAs and mRNAs from profile 16 and miRNAs identified in our previous study, which included 361 miRNAs, 68 circRNAs, 599 mRNAs, and 31,063 interacting pairs. Functional annotation showed that upregulated circRNAs might contribute to glycolysis/gluconeogenesis, biosynthesis of amino acids, pyruvate metabolism, carbon metabolism, glycogen and sucrose metabolism through the ceRNA network, and thus affected postnatal muscle development by regulating muscle protein deposition. Of them, circRNA225 and circRNA226 from the same host gene might be key circRNAs that could regulate muscle development by interacting with seven common miRNAs and 207 mRNAs. Our experiments also demonstrated that there were interactions among circRNA225, gga-miR-1306-5p, and heat shock protein alpha 8 (HSPA8).ConclusionsOur results suggest that adequate supply of nutrients such as energy and protein after hatching may be a key factor in ensuring chicken yield, and provide several candidate circRNAs for future studies concerning ceRNA regulation during chicken muscle development.

The Regulatory Network and Role of the circRNA-miRNA-mRNA ceRNA Network in the Progression and the Immune Response of Wilms Tumor Based on RNA-Seq.

Circular RNA (circRNA), which is a newly discovered non-coding RNA, has been documented to play important roles in miRNA sponges, and the dysregulation of which is involved in cancer development. However, circRNA expression profiles and their role in initiation and progression of Wilms tumor (WT) remain largely unclear at present. Here, we used paired WT samples and high-throughput RNA sequencing to identify differentially expressed circRNAs (DE-circRs) and mRNAs (DE-mRs). A total of 314 DE-circRs and 1612 DE-mRs were identified. The expression of a subset of differentially expressed genes was validated by qRT–PCR. A complete circRNA-miRNA-mRNA network was then constructed based on the common miRNA targets of DE-circRs and DE-mRs identified by miRanda prediction tool. The Gene set enrichment analysis (GSEA) indicated that several signaling pathways involving targeted DE-mRs within the ceRNA network were associated with cell cycle and immune response, which implies their participation in WT development to some extent. Subsequently, these targeted DE-mRs were subjected to implement PPI analysis and to identify 10 hub genes. Four hub genes were closely related to the survival of WT patients. We then filtered prognosis-related hub genes by Cox regression and least absolute shrinkage and selection operator (LASSO) regression analysis to construct a prognosis-related risk score system based on a three-gene signature, which showed good discrimination and predictive ability for WT patient survival. Additionally, we analyzed the mutational landscape of these genes and the associations between their expression levels and those of immune checkpoint molecules and further demonstrated their potential impact on the efficacy of immunotherapy. qRT–PCR and western blotting (WB) analysis were used to validate key differentially expressed molecules at the RNA and protein levels, respectively. Besides these, we selected a key circRNA, circEYA1, for function validation. Overall, the current study presents the full-scale expression profiles of circRNAs and the circRNA-related ceRNA network in WT for the first time, deepening our understanding of the roles and downstream regulatory mechanisms of circRNAs in WT development and progression. We further constructed a useful immune-related prognostic signature, which could improve clinical outcome prediction and guide individualized treatment.

Identification of Potential Key circRNAs in Aged Mice With Postoperative Delirium.

Postoperative delirium (POD) is a common postoperative complication in elderly patients and seriously affects postoperative recovery. The exact mechanism of POD is still unclear. Therefore, it is necessary to explore the mechanism of POD in transcriptional regulation. At present, circRNAs have been proven to play an important role in a variety of mental health and cognitive disorders, such as Alzheimer’s disease, depression and schizophrenia. To reveal the effect of circRNA on POD, we used microarray to analyze the differential expression profiles of circRNAs in the hippocampus of 12-month-old mice between the tibial fracture and control groups. A total of 1,4236 circRNAs were identified. Compared with the control group, there were 500 circRNAs with increased expression and 187 with decreased expression. The accuracy of the microarray data was further verified by qRT–PCR. Finally, GO enrichment and KEGG pathway analyses indicated that changes in axon orientation, ubiquitin-mediated proteolysis, glutamate synapses, the estrogen signaling pathway, the RAS signaling pathway and other systems may be important potential pathological mechanisms in the progression of POD. In particular, we found that the HOMER1 gene and its transcript mmu_circRNA_26701 are specifically expressed in the glutamate synapse, which may provide new clues and intervention targets for the progression of this refractory disease.

Identification of a circRNA-mediated comprehensive ceRNA network in spinal cord injury pathogenesis

RNAs are closely associated with human diseases; however, immune-related genes (IRGs) and their potential regulatory networks in relation to spinal cord injury (SCI) are still poorly understood. Here, we investigated the key IRGs as well as the competing endogenous RNA (ceRNA) mechanisms that are associated with SCI pathogenesis based on microarray datasets and the use of a rat SCI model. Specifically, four independent SCI microarray datasets from Gene Expression Omnibus (GEO) database were analyzed and, thereafter, differentially expressed IRGs were annotated via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Furthermore, based on the GEO datasets, differentially expressed RNAs (DERNAs), including DEcircRNAs, DEmiRNAs, and DEmRNAs were identified and interactions between them were also predicted using online databases, and to construct a circular RNA (circRNA) mediated ceRNA network, candidate RNAs were also identified. Furthermore, the support vector machine (SVM) and least absolute shrinkage and selection operator (LASSO) methods were used for the identification of critical DERNAs, while differential gene expression was validated using the GSE20907 dataset. Our results were as follows. In the SCI microarray datasets, 32, 58, and 74 DEIRGs, DEcircRNAs, and DEmiRNAs were identified, respectively. In addition, GO and KEGG analyses showed that the DEIRGs were primarily enriched in neutrophil-mediated immunity and nuclear factor-kappa B (NF-κB) and hypoxia-inducible factor-1 (HIF-1) signaling pathways, and based on LASSO and SVM screening, PLXNB2 was identified as a DEIRG, while hsa_circ_0026646 was identified as the key circRNA, showing a higher SCI expression. Furthermore, our results proved that PLXNB2 and hsa_circ_0026646 were upregulated in SCI, whereas miR-331-3p was downregulated, and, interestingly, similar expression profiles were confirmed using the rat SCI model. Furthermore, fluorescent reporter assay indicated that both hsa_circ_0026646 and PLXNB2 have miR-331-3p target sites, and the ceRNA hypothesis suggested the dysregulation of hsa_circ_0026646, miR-331-3p, and PLXNB2 in SCI. Thus, our results suggested that in SCI pathogenesis, hsa_circ_0026646 correlates with PLXNB2 by targeting miR-331-3p.

Identification of Key CircRNAs Related to Pulmonary Tuberculosis Based on Bioinformatics Analysis.

Pulmonary tuberculosis (TB) is a chronic infectious disease that is caused by respiratory infections, principally Mycobacterium tuberculosis. Increasingly, studies have shown that circular (circ)RNAs play regulatory roles in different diseases through different mechanisms. However, their roles and potential regulatory mechanisms in pulmonary TB remain unclear. In this study, we analyzed circRNA sequencing data from adjacent normal and diseased tissues from pulmonary TB patients and analyzed the differentially expressed genes. We then constructed machine learning models and used single-factor analysis to identify hub circRNAs. We downloaded the pulmonary TB micro (mi)RNA (GSE29190) and mRNA (GSE83456) gene expression datasets from the Gene Expression Omnibus database and performed differential expression analysis to determine the differentially expressed miRNAs and mRNAs. We also constructed a circRNA–miRNA–mRNA interaction network using Cytoscape. Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis were used to predict the biological functions of the identified RNAs and determine hub genes. Then, the STRING database and cytoHubba were used to construct protein-protein interaction networks. The results showed 125 differentially expressed circRNAs in the adjacent normal and diseased tissues of pulmonary TB patients. Among them, we identified three hub genes associated with the development of pulmonary TB: hsa_circ_0007919 (upregulated), hsa_circ_0002419 (downregulated), and hsa_circ_0005521 (downregulated). Through further screening, we determined 16 mRNAs of potential downstream genes for hsa-miR-409-5p and hsa_circ_0005521 and established an interaction network. This network may have important roles in the occurrence and development of pulmonary TB. We constructed a model with 100% prediction accuracy by machine learning and single-factor analysis. We constructed a protein-protein interaction network among the top 50 hub mRNAs, with FBXW7 scoring the highest and SOCS3 the second highest. These results may provide a new reference for the identification of candidate markers for the early diagnosis and treatment of pulmonary TB.

A microarray expression profile and bioinformatic analysis of circular RNA in human esophageal carcinoma

Recent studies indicate that non-coding circular RNAs (circRNAs) are involved in the development of esophageal carcinoma (EC). This study aimed to identify differential expression of circRNAs in EC, which can provide potential biomarkers and therapeutic targets for EC treatment and improve the understanding of tumorigenesis mechanism. First, samples (n=5) of EC tissues and adjacent normal tissue were sent for circRNA microarray detection, Second, further bioinformatic analysis was performed, including circRNA-microRNA (miRNA), co-expression network analysis, Spearman correlation test, and cancer-related circRNA-miRNA axis analysis. Finally, the expression of circRNA that our analysis predicted to be hub genes was verified in samples (n=15) of EC tissues and adjacent normal tissue by real-time polymerase chain reaction (RT-PCR). Microarray identified 102 upregulated and 67 significantly downregulated circRNAs were in EC patients' tumors relative to adjacent normal tissue. One upregulated circRNA (hsa_circRNA_401955) showed the most connection with MREs, therefore was regarded as the hub gene by the Spearman correlation test. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that four primary pathways (mRNA surveillance, cytoskeleton actin regulation, spliceosome, and the NOD-like receptor signaling pathway) were predicted in the hub circRNA's five connected miRNA response elements (MREs). Furthermore, cancer-related circRNA-miRNA axis analyses showed that hsa_circRNA_100375 and its four connected MREs participated in the cancer-related pathway. RT-PCR showed that hsa_circRNA_100375 and hsa_circRNA_401955 were significantly increased in the tumor tissues of EC patients. Abnormal expression of circRNAs was involved in the tumorigenesis of EC. Key circRNAs, namely hsa_circRNA_401955 and hsa_circRNA_100375, may be as potential biomarkers and therapeutic targets for the treatment of EC.

Construction and Bioinformatics Analysis of circRNA-miRNA-mRNA Network in Acute Myocardial Infarction.

Background: Acute myocardial infarction (AMI) is one of the main fatal diseases of cardiovascular diseases. Circular RNA (circRNA) is a non-coding RNA (ncRNA), which plays a role in cardiovascular disease as a competitive endogenous RNA (ceRNA). However, their role in AMI has not been fully clarified. This study aims to explore the mechanism of circRNA-related ceRNA network in AMI, and to identify the corresponding immune infiltration characteristics. Materials and Methods: The circRNA (GSE160717), miRNA (GSE24548), and mRNA (GSE60993) microarray datasets of AMI were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed circRNAs (DEcircRNAs), miRNAs (DEmiRNAs), and mRNAs (DEmRNAs) were identified by the “limma” package. After integrating the circRNA, miRNA and mRNA interaction, we constructed a circRNA-miRNA-mRNA network. The “clusterProfiler” package and String database were used for functional enrichment analysis and protein-protein interaction (PPI) analysis, respectively. After that, we constructed a circRNA-miRNA-hub gene network and validated the circRNAs and mRNAs using an independent dataset (GSE61144) as well as qRT-PCR. Finally, we used CIBERSORTx database to analyze the immune infiltration characteristics of AMI and the correlation between hub genes and immune cells. Results: Using the “limma” package of the R, 83 DEcircRNAs, 54 DEmiRNAs, and 754 DEmRNAs were identified in the microarray datasets of AMI. Among 83 DEcircRNAs, there are 55 exonic DEcircRNAs. Then, a circRNA-miRNA-mRNA network consists of 21 DEcircRNAs, 11 DEmiRNAs, and 106 DEmRNAs were predicted by the database. After that, 10 hub genes from the PPI network were identified. Then, a new circRNA-miRNA-hub gene network consists of 14 DEcircRNAs, 7 DEmiRNAs, and 9 DEmRNAs was constructed. After that, three key circRNAs (hsa_circ_0009018, hsa_circ_0030569 and hsa_circ_0031017) and three hub genes (BCL6, PTGS2 and PTEN) were identified from the network by qRT-PCR. Finally, immune infiltration analysis showed that hub genes were significantly positively correlated with up-regulated immune cells (neutrophils, macrophages and plasma cells) in AMI. Conclusion: Our study constructed a circRNA-related ceRNA networks in AMI, consists of hsa_circ_0031017/hsa-miR-142-5p/PTEN axis, hsa_circ_0030569/hsa-miR-545/PTGS2 axis and hsa_circ_0009018/hsa-miR-139-3p/BCL6 axis. These three hub genes were significantly positively correlated with up-regulated immune cells (neutrophils, macrophages and plasma cells) in AMI. It helps improve understanding of AMI mechanism and provides future potential therapeutic targets.

Redondoviridae infection regulates circRNAome in periodontitis.

Redondoviridae is a recently identified family of DNA viruses associated with periodontitis. Circular RNAs (circRNAs) are novel endogenous, conserved noncoding RNAs contributing to the virus-related immune-inflammatory response. This study aimed to analyze the expression profiles of circRNAs in the gingival tissues of periodontitis patients with and without Redondoviridae-infection and healthy controls using high-throughput RNA sequencing combined with experimental validation. Out of 17 819 circRNAs, 175 were dysregulated. Functional annotation and enrichment analysis of the differential circRNA host genes demonstrated potential alterations in the molecular and cellular components and metabolism in individuals suffering from periodontitis with Redondoviridae infection. Moreover, "axon guidance," "lysine biosynthesis," and "vascular endothelial growth factor signaling pathways" were significantly enriched in Redondoviridae-infected gingivitis tissues. Furthermore, the key circRNAs (circCOL1A1, circAASS, circPTK2, circATP2B4, circDOCK1, circTTBK2, and circMCTP2) associated with the pathobiology of Redondoviridae-related periodontitis were identified by constructing circRNA-micro RNA (miRNA)-messenger RNA (mRNA) networks. Bioinformatics analyses demonstrated that abnormally expressed circRNAs might contribute to the etiopathogenesis and development of Redondoviridae-related periodontitis. This study's findings have enhanced the current understanding of the Redondoviridae-related periodontitis mechanism and provide insights into further applications for diagnostic markers and therapeutic uses.

Expression Profile Analysis to Identify Circular RNA Expression Signatures in the Prolificacy Trait of Yunshang Black Goat Pituitary in the Estrus Cycle.

The pituitary gland is an important organ. It is a complex area of the brain involved in endocrine function and reproductive regulation. However, the function of the pituitary in goat reproduction is still unclear. Herein, RNA sequencing was used to explore the expression patterns of circle RNAs (circRNAs) in the pituitary of Yunshang black goats during the various estrus phases. Then the host genes of the circRNAs were predicted, and a competing endogenous RNA (ceRNA) network was constructed. The results showed a total of 6,705 circRNAs in the pituitary of Yunshang black goats, among which 388 differentially expressed (DE) circRNAs (214 were upregulated, while 174 were downregulated) were identified between high- and low-yield Yunshang black goats in the follicular phase (HF vs. LF); moreover, 361 DE circRNAs (136 were upregulated, while 225 were downregulated) were identified between high- and low-yield Yunshang black goats in the luteal phase (HL vs. LL). There were 65 DE circRNAs targeting 40 miRNAs in the HF vs. LF comparison and 46 DE circRNAs targeting 31 miRNAs in the HL vs. LL comparison. We identified chi_circ_0030920, chi_circ_0043017, chi_circ_0008353, chi_circ_0041580, and chi_circ_0016478 as the key circRNAs through functional enrichment analysis. The ceRNA network analysis showed that chi_circ_0031209 and chi_circ_0019448 might play an important role in reproduction by influencing the expression of prolactin receptor (PRLR) in high- and low-yielding goats during the luteal phase, whereas chi_circ_0014542 regulates the expression of WNT5A during the follicular phase. Our study provided the overall expression profiles of circRNAs in the goat pituitary during the estrus phase, which provides new insight into the mechanism of high-yield goats, which can be helpful to guide goat breeding.

Hsa_Circ_0001860 Promotes Smad7 to Enhance MPA Resistance in Endometrial Cancer via miR-520h.

Background: Medroxyprogesterone acetate (MPA) is one of the most commonly prescribed progestin for the treatment of endometrial cancer (EC). Despite initial benefits, many patients ultimately develop progesterone resistance. Circular RNA (circRNA) is a kind of noncoding RNA, contributing greatly to the development of human tumor. However, the role of circular RNA in MPA resistance is unknown. Methods: We explored the expression profile of circRNAs in Ishikawa cells treated with (ISK/MPA) or without MPA (ISK) by RNA sequencing, and identified a key circRNA, hsa_circ_0001860. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to verify its expression in MPA-resistant cell lines and tissues. CCK8, Transwell, and flow cytometry were used to evaluate the functional roles of hsa_circ_0001860 in MPA resistance. The interaction between hsa_circ_0001860 and miR-520 h was confirmed by bioinformatics analysis, luciferase reporter assay, and RNA pull-down assay. Results: The expression of hsa_circ_0001860 was significantly downregulated in MPA-resistant cell lines and tissues, and negatively correlated with lymph node metastasis and histological grade of EC. Functional analysis showed that hsa_circ_0001860 knockdown by short hairpin RNA (shRNA) promoted the proliferation, inhibited the apoptosis of Ishikawa cells, and promoted the migration and invasion of Ishikawa cells treated with MPA. Mechanistically, hsa_circ_0001860 promoted Smad7 expression by sponging miR-520 h. Conclusion: Hsa_circ_0001860 plays an important role in the development of MPA resistance in EC through miR-520h/Smad7 axis, and it could be targeted to reverse the MPA resistance in endometrial cancer.

Emerging roles of circRNAs in regulating thermal and hypoxic stresses in Apostichopus japonicus (Echinodermata: Holothuroidea)

Some sea cucumbers are economically and ecologically important, but they are threatened by thermal and hypoxic stress in changing oceanographic conditions. We construct circRNAs profiles, reveal circRNAs characters, and illustrate the potential regulatory roles of circRNAs in one commercially important species of sea cucumber, Apostichopus japonicus. Reads are distributed in intergenic (44.14%), exonic (48.26%) and intronic (7.60%) regions of the genome. A total of 1684 circRNAs were identified, and the most common spliced length is 269 nt in the present study. In three treatments (HT [thermal stress], LO [hypoxic stress], and HL [combined thermal and hypoxic stress]), 24, 27 and 27 differentially expressed (DE) circRNAs were identified, respectively. Five novel DE-circRNAs commonly occur in these treatments (novel_circ_0003311, novel_circ_0000229, novel_circ_0003944, novel_circ_0001458 and novel_circ_0000707), and based on them, potential circRNA-miRNA binding pairs were predicted. Sanger sequencing, RNase R treatment experiment and qPCR validation identified the accuracy of the circRNAs. Key circRNAs identified in the present study were covalently closed and were more stable under RNase R treatment than linear RNAs. Based on function analysis, circRNAs could regulate metabolic process, signal transduction, and ion responses in A. japonicus when exposed to thermal and hypoxic stress, and ‘regulation of response to stimulus’ is a common gene ontology (GO) term that is significantly enriched in each treatment; GO terms for ‘DNA’ and ‘stress’ are commonly enriched in heat-related treatments (HT and HL); and GO terms for ‘protein’ are commonly enriched in hypoxia-related treatments (LO and HL). When environmentally stressed, ‘metabolism,’ ‘transport and catabolism,’ ‘membrane transport,’ and ‘signal transduction’ were significantly responded in sea cucumber based on KEGG analysis. We provide insights into circRNA functions in stress regulation and lay a foundation for invertebrate circRNA research.

The potential role of hsa_circ_0001079 in androgenetic alopecia via sponging hsa-miR-136-5p.

BackgroundAndrogenetic alopecia (AGA) is an androgen‐dependent polygenic hereditary disease.MethodsDiseased hair follicles from 5 AGA patients and normal hair follicles from 5 healthy individuals were selected as specimens to carry out whole transcriptome sequencing. Multiple high‐expression circular RNAs (circRNAs) were screened from the diseased group. We further verified the presence of the circRNAs in the clinical specimens by real‐time fluorescence quantitative PCR (qRT‐PCR).ResultsIn total, 100 circRNAs were significantly upregulated, and 184 circRNAs were significantly downregulated. The top 10 upregulated circRNAs were hsa_circ_0101041, hsa_circ_0001578, hsa_circ_0135062, hsa_circ_0002980, hsa_circ_0005062, hsa_circ_0072688, hsa_circ_0133954, hsa_circ_0001079, hsa_circ_0005974, hsa_circ_0000489. The top 10 downregulated circRNAs were hsa_circ_0001278, hsa_circ_0031482, hsa_circ_0008285, hsa_circ_0003784, hsa_circ_0077096, hsa_circ_0001148, hsa_circ_0006886, hsa_circ_0000638, hsa_circ_0084521, and hsa_circ_0101074. Among top 10 upregulated circRNAs, hsa_circ_0001079 showed significantly high expression via large‐sample verification and clinical application potential. Based on a database comparison and base pairing analysis, we found that has‐miR‐136‐5p bound to hsa_circ_0001079 and that hsa‐miR‐136‐5p had potential binding sites with Wnt5A.ConclusionIn summary, through high‐throughput sequencing and bioinformatic analysis, a potential diagnostic marker for alopecia and a key circRNA that might adsorb microRNA (miRNA) through a sponging mechanism, thus mediating alopecia, were discovered in this study.

Whole transcriptome sequencing identifies key circRNAs, lncRNAs, and miRNAs regulating neurogenesis in developing mouse retina

BackgroundThe molecular complexity of neural retina development remains poorly studied. Knowledge of retinal neurogenesis regulation sheds light on retinal degeneration therapy exploration. Therefore, we integrated the time-series circRNA, lncRNA, miRNA, and mRNA expression profiles of the developing retina through whole-transcriptome sequencing. The key functional ncRNAs and the ceRNA network regulating retinal neurogenesis were identified.ResultsTranscriptomic analysis identified circRNA as the most variable ncRNA subtype. We screened a series of neurogenesis-related circRNAs, lncRNAs, and miRNAs using different strategies based on their diversified molecular functions. The expression of circCDYL, circATXN1, circDYM, circPRGRIP, lncRNA Meg3, and lncRNA Vax2os was validated by quantitative real-time PCR. These circRNAs and lncRNAs participate in neurotransmitter transport and multicellular organism growth through the intricate circRNA/lncRNA-miRNA-mRNA network.ConclusionWhole-transcriptome sequencing and bioinformatics analysis systematically screened key ncRNAs in retinal neurogenesis. The validated ncRNAs and their circRNA/lncRNA-miRNA-mRNA network involve neurotransmitter transport and multicellular organism growth during retinal development.

Screening for differentially expressed circRNAs in ischemic stroke by RNA sequencing

BackgroundIschemic stroke is a disease with high rate of death and disability worldwide. CircRNAs, as a novel type of non-coding RNAs, lacking 5’ caps and 3’ poly-A tails, has been associated with ischemic stroke. This study aimed to investigate key circRNAs related to ischemic stroke.MethodsRNA sequencing was performed obtain the circRNA expression profiles from peripheral whole blood of three ischemic stroke patients and three healthy individuals. Through bioinformatic analysis, differentially expressed circRNAs (DEcircRNAs) were identified, and GO and pathway analyses for the host genes of DEcircRNAs were conducted. The expression levels of selected circRNAs were analyzed with qRT-PCR. To further explore the functions of key circRNAs, a DEcircRNA-miRNA interaction network was constructed.ResultsA total of 736 DEcircRNAs were detected in ischemic stroke. Functional annotation of host genes of DEcircRNAs revealed several significantly enriched pathways, including Fc epsilon RI signaling pathway, B cell receptor signaling pathway, and T cell receptor signaling pathway. The qRT-PCR results were largely in keeping with our RNA-seq data. The ROC curve analyses indicated that hsa_circ_0000745, hsa_circ_0001459, hsa_circ_0003694 and hsa_circ_0007706 with relatively high diagnostic value. A circRNA-miRNA network, including 1544 circRNA-miRNA pairs, 456 circRNAs and 4 miRNAs, was obtained.ConclusionsThe results of our study may help to elucidate the specific mechanism underlying ischemic stroke.

Circular RNA Cwc27 contributes to Alzheimer's disease pathogenesis by repressing Pur-α activity.

Circular RNAs (circRNAs) have gained growing attention in participating in various biological processes and referring to multiply kinds of diseases. Although differentially expressed circRNA profiling in Alzheimer's disease (AD) has been established, little is known about the precise characteristic and functions of key circRNAs with direct relevance to AD in gene expression and disease-related cognition. Herein, we screened and identified circCwc27 as a novel circRNA implicated in AD. CircCwc27 was a neuronal-enriched circRNA that abundantly expressed in the brain and significantly upregulated in AD mice and patients. Knockdown of circCwc27 markedly improved AD-related pathological traits and ameliorated cognitive dysfunctions. Mechanistically, we excluded the miRNA decoy mechanism and focused on the important function of circRNA-RNA-binding protein (RBP) interaction in AD. CircCwc27 directly bound to purine-rich element-binding protein A (Pur-α), increased retention of cytoplasmic Pur-α, and suppressed Pur-α recruitment to the promoters of a cluster of AD genes, including amyloid precursor protein (APP), dopamine receptor D1 (Drd1), protein phosphatase 1, regulatory inhibitor subunit1B (Ppp1r1b), neurotrophic tyrosine kinase, receptor, type 1 (Ntrk1), and LIM homeobox 8 (Lhx8). Downregulation of circCwc27 enhanced the affinity of Pur-α binding to these promoters, leading to altered transcription of Pur-α targets. Moreover, Pur-α overexpression largely phenocopied circCwc27 knockdown in preventing Aβ deposition and cognitive decline. Together, our findings suggest significant functional consequences of a circRNA-protein interaction, that circCwc27, by associating with the regulatory protein Pur-α, may act as a crucial player in AD pathogenesis and represent a promising AD therapeutic target with clinical translational potential.