Identification Of Circular RNAs Research Articles

Role of circular RNA/miRNA axes in the pathophysiology of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a disorder resulted from interactions between genetic and environmental factors. Based on the importance of epigenetic factors in the pathoetiology of PCOS, the current review focused on identification of circular RNAs (circRNAs) that are involved in PCOS through acting as molecular sponges for microRNAs (miRNAs). The literature search led to identification of circ_0043533/miR-1179, circ_0030018/miR-136, circ_FURIN/miR-423-5p, circ-FURIN/miR-195-5p, circ_0043532/miR-182, circ_RANBP9/miR-136-5p, circRHBG/miR-515-5p, circMTO1/miR-320b, circASPH/miR-375, circPSMC3/miR-296-3p, circLDLR/miR-1294, circPUM1/miR-760, and hsa_circ_0118530/miR-136 as molecular axes contributing to the pathogenesis of PCOS. To set the stage for future research on the role of the ceRNA network in PCOS, in-silico analyses were performed using miRWalk, miRNet, and miRDIP databases. miRWalk identified 80 genes regulated by 5 miRNAs, miRNet revealed 6449 circRNAs potentially controlling 11 miRNAs, and miRDIP identified 11 miRNAs associated with 35 human pathways. These targets can be used in the treatment options, design of personalized medicine and prediction of prognosis of PCOS.

Circular mitochondrial-encoded mRNAs are a distinct subpopulation of mitochondrial mRNA in Trypanosoma brucei

Since the first identification of circular RNA (circRNA) in viral-like systems, reports of circRNAs and their functions in various organisms, cell types, and organelles have greatly expanded. Here, we report the first evidence, to our knowledge, of circular mRNA in the mitochondrion of the eukaryotic parasite, Trypanosoma brucei. While using a circular RT-PCR technique developed to sequence mRNA tails of mitochondrial transcripts, we found that some mRNAs are circularized without an in vitro circularization step normally required to produce PCR products. Starting from total in vitro circularized RNA and in vivo circRNA, we high-throughput sequenced three transcripts from the 3′ end of the coding region, through the 3′ tail, to the 5′ start of the coding region. We found that fewer reads in the circRNA libraries contained tails than in the total RNA libraries. When tails were present on circRNAs, they were shorter and less adenine-rich than the total population of RNA tails of the same transcript. Additionally, using hidden Markov modelling we determined that enzymatic activity during tail addition is different for circRNAs than for total RNA. Lastly, circRNA UTRs tended to be shorter and more variable than those of the same transcript sequenced from total RNA. We propose a revised model of Trypanosome mitochondrial tail addition, in which a fraction of mRNAs is circularized prior to the addition of adenine-rich tails and may act as a new regulatory molecule or in a degradation pathway.

Identification of circular RNAs in tea plant during Helopeltis theivora infestation

The tea mosquito bug (TMB) or Helopeltis theivora has been reported as a significant tea pest that causes major tea crop loss. Circular RNAs (circRNAs) are a newly discovered group of the non-coding RNA (ncRNA) and are known to function in gene regulation process. Tea plant (Camellia sinensis) has not been explored for the detection of circRNAs during pest infestation. This is the first report of circRNAs identified in C. sinensis infested by a sap-sucking insect H. theivora. In total, we identified 709 circRNAs in six RNA-seq libraries. Analysis of the differential expression pattern revealed that 34 circRNAs got upregulated/downregulated in response to H. theivora attack. Functional annotation and enrichment analysis of the differentially expressed circRNAs showed that the circRNA-target genes were enriched in biological pathways like “tryptophan metabolism”, “biosynthesis of secondary metabolites”, “porphyrin metabolism”, “nucleocytoplasmic transport”, “N-glycan biosynthesis” etc. miRNA-mediated interaction analysis of the circRNAs also showed that 17 differentially expressed circRNAs could also act as “sponges” thus masking the miRNA-mRNA binding and subsequently repressing mRNA cleavage. These differentially expressed circRNAs were predicted to have the capacity to behave as endogenous target mimics (eTMs) for potential genes like aspartyl protease, phospholipase, lectin receptor etc.

Identification of circular RNAs as regulators and noninvasive biomarkers for placenta accreta spectrum.

Placenta accreta spectrum (PAS) has an urgent need for reliable prenatal biomarkers. This study profiled the circular RNAs (circRNAs) in PAS placenta and maternal blood and identified two circRNAs can regulate trophoblast cells invasion and serve as noninvasive prenatal biomarkers for PAS prediction. PAS is one of the most alarming obstetric diseases with high mortality rates. The regulating mechanism underlying PAS remains to be investigated, and reliable blood biomarkers for PAS have not emerged. Circular RNAs (circRNAs) have become important regulators and biomarkers for disparate human diseases. However, the circRNA profiles of PAS were not reported, and the regulatory role and predictive value of circRNAs in PAS were unknown. Here, we comprehensively profiled the circRNAs in the placenta of PAS by transcriptome sequencing and analysis and uncovered 217 abnormally expressed circRNAs. Through competing endogenous RNA network analysis, we found that the target genes of upregulated circRNAs in PAS were enriched in placenta development-related pathways and further uncovered two circRNAs, circPHACTR4 and circZMYM4, that could regulate trophoblast cells invasion and migration in vitro. Finally, we verified that circPHACTR4 and circZMYM4 were also upregulated in the maternal peripheral blood of PAS women before delivery using transcriptome sequencing and RT-qPCR and evaluated their predictive value by ROC curves. We found that circPHACTR4 and circZMYM4 could serve as effective predicting biomarkers for PAS (area under the curve (AUC): 0.86 and 0.85) and propose an improved model for PAS prenatal prediction by combining the conventional ultrasound diagnosis with the new circRNA predictive factors (AUC: 0.91, specificity: 0.89, sensitivity: 0.82).Altogether, this work provides new resources for deciphering the biological roles of circRNAs in PAS, identified two circRNAs that could regulate trophoblast cells invasion during placentation, and revealed two noninvasive diagnostic markers for PAS.

Identification of circular RNAs in resistant tomato genotype in response to ToLCBaV infection

Circular RNAs (CircRNAs) are covalently closed non-coding RNAs that play an important role in a variety of biological processes. CircRNA profiling helps to understand biological process associated with various abiotic and biotic stresses. In tomato genotype IIHR- 2611 (resistant to ToLCBaV), a total of 193 CircRNAs were discovered, of which 72 and 121 were found in control (RC) and ToLCBaV inoculated (RI) plants respectively. Among them, 103 (53 %) were exonic CircRNA regulating the expressions of their parent genes. Relative expression of CircRNAs 2:45295638|45295796, 2:51520741|51530067 and 7:67566489|67566691 and their respective parent genes Solyc02g080530.3 (peroxidase), Solyc02g088950.2 (superoxide dismutase) and Solyc07g065840.2.1 (heat shock protein 90) response to ToLCBaV infection were analysed at different time intervals. A significantly positive correlation was observed for the expression profiles of all three circRNAs and their parent genes. Furthermore, the differential expression across samples as well as time interval indicates that CircRNA mediated gene expression is involved in viral resistance. The results of the expression assays of both superoxide dismutase and peroxidase were consistent with enzyme analysis. Overall findings demonstrated the importance of CircRNAs in ToLCBaVD resistance and suggested that CircRNAs could be key regulators of gene expression during disease resistance in tomato.

CircRNA-regulated immune responses of asian honey bee workers to microsporidian infection.

Nosema ceranae is a widespread fungal parasite for honey bees, causing bee nosemosis. Based on deep sequencing and bioinformatics, identification of circular RNAs (circRNAs) in Apis cerana workers’ midguts and circRNA-regulated immune response of host to N. ceranae invasion were conducted in this current work, followed by molecular verification of back-splicing sites and expression trends of circRNAs. Here, 10185 and 7405 circRNAs were identified in the midguts of workers at 7 days (AcT1) and 10 days (AcT2) post inoculation days post-inoculation with N. ceranae. PCR amplification result verified the back-splicing sites within three specific circRNAs (novel_circ_005123, novel_circ_007177, and novel_circ_015140) expressed in N. ceranae-inoculated midgut. In combination with transcriptome data from corresponding un-inoculated midguts (AcCK1 and AcCK2), 2266 circRNAs were found to be shared by the aforementioned four groups, whereas the numbers of specific ones were 2618, 1917, 5691, and 3723 respectively. Further, 83 52) differentially expressed circRNAs (DEcircRNAs) were identified in AcCK1 vs. AcT1 (AcCK2 vs. AcT2) comparison group. Source genes of DEcircRNAs in workers’ midgut at seven dpi were involved in two cellular immune-related pathways such as endocytosis and ubiquitin mediated proteolysis. Additionally, competing endogenous RNA (ceRNA) network analysis showed that 23 13) DEcircRNAs in AcCK1 vs. AcT1 (AcCK2 vs. AcT2) comparison group could target 18 14) miRNAs and further link to 1111 (1093) mRNAs. These target mRNAs were annotated to six cellular immunity pathways including endocytosis, lysosome, phagosome, ubiquitin mediated proteolysis, metabolism of xenobiotics by cytochrome P450, and insect hormone biosynthesis. Moreover, 284 164) internal ribosome entry site and 54 26) ORFs were identified from DEcircRNAs in AcCK1 vs. AcT1 (AcCK2 vs. AcT2) comparison group; additionally, ORFs in DEcircRNAs in midgut at seven dpi with N. ceranae were associated with several cellular immune pathways including endocytosis and ubiquitin-mediated proteolysis. Ultimately, RT-qPCR results showed that the expression trends of six DEcircRNAs were consistent with those in transcriptome data. These results demonstrated that N. ceranae altered the expression pattern of circRNAs in A. c. cerana workers’ midguts, and DEcircRNAs were likely to regulate host cellular and humoral immune response to microsporidian infection. Our findings lay a foundation for clarifying the mechanism underlying host immune response to N. ceranae infection and provide a new insight into interaction between Asian honey bee and microsporidian.

Identification of Circular RNAs in the Ovaries of Hu Sheep and Local Fat-tailed Sheep during the Luteal Phase

Background: Circular RNAs (circRNAs), a novel class of endogenous non-coding RNAs with covalently closed loop structures, play important role in mammalian growth and development by regulating gene expression and participating in various biological processes. The aim of this study was to identify the expression profiles of circRNAs in the ovaries of Hu sheep and local Fat-tailed sheep during the luteal phase to determine the molecular mechanisms underlying the effects of circRNAs on reproduction and follicular development in sheep. Methods: To identify the differentially expressed circRNAs expressed in luteal phase conditions in Hu sheep and fat-tailed sheep, ovarian samples collected during the luteal phase were analyzed using high-throughput sequencing and subsequent transcriptomic approaches and bioinformatics analysis. Result: A total of 2,875 differentially expressed circRNAs were identified by high-through sequencing. Gene Ontology terminology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the host genes were mainly involved in the secretion and metabolism of the various pathway. Quantitative real-time PCR was performed to validate the results of expression profiling. These results contributed to an improved understanding of circRNA and the biological mechanisms of ovarian reproduction in sheep.

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.

Identification of circular RNA_0000919 as a potential diagnostic and prognostic biomarker of tongue squamous cell carcinoma using circular RNA microarray and reverse transcription-quantitative PCR analyses.

The present study aimed to identify differentially expressed (DE) circular RNAs (circRNAs/circs) using microarray analysis and to further explore the clinical significance of 10 candidate DEcircRNAs in patients with tongue squamous cell carcinoma (TSCC). A total of 60 patients with TSCC who underwent surgery were enrolled and five pairs of TSCC and adjacent (Ctrl) tissues were used for circRNA microarray analysis. Subsequently, the top five upregulated and downregulated DEcircRNAs were detected by reverse transcription-quantitative PCR (RT-qPCR) analysis in 60 pairs of tumor and Ctrl tissues, and their association with tumor features and overall survival (OS) was further analyzed. circRNA expression was used to differentiate TSCC from Ctrl tissues by principal component and heatmap analyses. A total of 134 upregulated and 67 downregulated DEcircRNAs were identified in TSCC tissues compared with Ctrl tissues. The DEcircRNAs were enriched in oncogenic signaling, including the ‘Wnt signaling pathway’ and the ‘MAPK signaling pathway’. The majority of DEcircRNAs exhibited several target microRNAs (miRNAs) in regulatory network analysis. These findings were validated by RT-qPCR analysis and the results demonstrated that the expression levels of 9/10 selected candidate DEcircRNAs (circ_0020048, circ_0000919, circ_0004525, circ_0002113, circ_0004029, circ_0004503, circ_0008752, circ_0002300 and circ_0001811) were dysregulated in TSCC tissues compared with Ctrl tissues. The expression levels of five DEcircRNAs (circ_0004503, circ_0008752, circ_0002300, circ_0020048 and circ_0000919) were associated with pathological grade or tumor clinical stage. Notably, only the expression levels of one DEcircRNA (circ_0000919) were associated with decreased OS. In conclusion, the present study indicated aberrant circRNA expression and potential circRNA-miRNA interactions in TSCC and identified circ_0000919 as a diagnostic and prognostic biomarker for TSCC management.

Cost-Effective Transcriptome-Wide Profiling of Circular RNAs by the Improved-tdMDA-NGS Method.

Covalently closed circular RNAs are neoteric to the eukaryotic family of long non-coding RNAs emerging as a result of 5′–3′ backsplicing from exonic, intronic, or intergenic regions spanning the parental gene. Owing to their unique structure and stability, circular RNAs have a multitude of functional properties such as micro-RNA and protein sponges, direct and indirect modulators of gene expression, protein translation, and many unproven activities apart from being potential biomarkers. However, due to their low abundance, most of the global circular RNA identification is carried out by high-throughput NGS-based approaches requiring millions of sequencing reads. This lag in methodological advancements demands for newer, more refined, and efficient identification techniques. Here, we aim to show an improved version of our previously reported template-dependent multiple displacement amplification (tdMDA)-NGS method by superimposing the ribosomal depletion step and use of H minus reverse transcriptase and RNase H. Implication of tdMDA using highly replicative Phi29 DNA polymerase after minimizing the linear and ribosomal RNA content further intensifies its detection limit toward even the abysmally expressing circular RNA at a low NGS depth, thereby decreasing the cost of identifying a single circular RNA. A >11-fold and >6-fold increase in total circular RNA was identified from the improved-tdMDA-NGS method over the traditional method of circRNA sequencing using DCC and CIRI2 pipelines, respectively, from Oryza sativa subsp. Indica. Furthermore, the reliability of the improved-tdMDA-NGS method was also asserted in HeLa cell lines, showing a significant fold difference in comparison with the existing traditional method of circRNA sequencing. Among the identified circular RNAs, a significant percentage from both rice (∼58%) and HeLa cell lines (∼84%) is found to be matched with the previously reported circular RNAs, suggesting that the improved-tdMDA-NGS method can be adapted to detect and characterize the circular RNAs from different biological systems.

Identification of circular RNAs and functional competing endogenous RNA networks in human proximal tubular epithelial cells treated with sodium-glucose cotransporter 2 inhibitor dapagliflozin in diabetic kidney disease

ABSTRACT Diabetic kidney disease (DKD) is a serious diabetes complication. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are novel anti-diabetes drugs that confer clinical renal protection. However, the molecular mechanisms involved remain unclear. Here, human proximal tubular epithelial cells (PTECs) were treated with normal glucose, high glucose, and anti-diabetes agents, including SGLT2i (dapagliflozin), metformin, and dipeptidyl peptidase-4 inhibitor (DPP-4i, vildagliptin) and microarray analysis was performed. Firstly, a total of 2,710 differentially expressed circular RNAs (circRNAs) were identified. Secondly, network pharmacology and transcriptomics analyses showed that the effects of dapagliflozin on PTECs primarily involved lipid metabolism, Rap1, and MAPK signaling pathways. Metformin mainly affected the AMPK and FOXO signaling pathways, whereas vildagliptin affected insulin secretion and the HIF-1 signaling pathway. Furthermore, circRNA-miRNA-mRNA networks, real-time reverse transcription-polymerase chain reaction (RT-PCR), and fluorescence in situ hybridization (FISH) assay revealed that the expression of hsa_circRNA_012448 was increased in PTECs treated with high glucose, whereas its expression was reversed by dapagliflozin. Finally, the hsa_circRNA_012448-hsa-miR-29b-2-5p-GSK3β pathway, involved in the oxidative stress response, was identified as an important pathway mediating the action of dapagliflozin against DKD. Overall, our study provides novel insights into the molecular mechanisms underlying the effects of dapagliflozin on DKD.

Identification of Circular RNA hsa-circ-0006969 as a Novel Biomarker for Breast Cancer

Background: To investigate the characteristics of circular RNA hsa-circ-0006969 in breast cancer and identify it as a novel biomarker for breast cancer. Methods: Three breast cancer (BC) patient tissues were selected to perform human circRNA microarray analysis. GeneSpring 13.0 (Agilent) software was applied for analyzing the data. Another 116 BC patients were recruited for verification. Hsa-circ-0006969 was found as a potential circRNA for BC diagnostic biomarker. The structure of hsa-circ-0006969 was predicted by circPrimer1.2 software. MiRanda v3.3, RNA hybrid 2.1, and Cytoscape 3.6.0 were used for predicting the networks of circRNA-miRNA. T-test, Curve regression, and ROC analysis were applied to certify the diagnostic values of hsa-circ-0006969. Using SPSS25.0 software to perform the Statistical analysis. Results: 546 higher expression and 1475 lower expression circRNAs were identified. Four circRNAs were filtrated for further verification. Hsa-circ-0006969 was significantly low expressed in BC tissues and peripheral blood. Hsa-circ-0006969 was confirmed as higher diagnostic correlation with BC tissues (AUC = 0.965) and peripheral blood (AUC = 0.842), with Grade 1 (AUC = 0.639), ER-positive (AUC = 0.612), TNM I (AUC = 0.693), TNM II (AUC = 0.712), TNM III (AUC = 0.757), TNM early stage(I, II) (AUC = 0.709). Hsa-circ-0006969 presented more effective diagnostic values for tumor metastasis (AUC = 0.784) compared with CA153 (AUC = 0.752). Conclusion: Hsa-circ-0006969 could be a novel biomarker for the diagnosis and treatment of BC.

Identification of circular RNAs and corresponding regulatory networks reveals potential roles in the brains of honey bee workers exposed to dinotefuran

Honey bees are important and highly efficient pollinators of agricultural crops and have been negatively affected by insecticides in recent years. Circular RNA (circRNA) plays an important role in the regulation of multiple biological and pathological processes; however, its role in the honey bee brain after exposure to dinotefuran is not well understood. Here, the expression profiles and potential modulation networks of circRNAs in the brains of workers (Apis mellifera) were comprehensively investigated using RNA sequencing and bioinformatics. In total, 33, 144, and 211 differentially expressed (DE) circRNAs were identified on the 1st, 5th and 10th days after exposure to dinotefuran, respectively. Enrichment analyses revealed that the host genes of DE circRNAs were enriched in the Hippo signaling pathway-fly, Wnt signaling pathway, and neuroactive ligand-receptor interaction. circ_0002266, circ_0005080, circ_0010239 and circ_0005415 were found to have translational potential due to the presence of an internal ribosome entry site (IRES). An integrated analysis of the DE circRNA-miRNA-mRNA networks suggest that circ_0008898 and circ_0001829 may participate in the immune response to dinotefuran exposure by acting as miRNA sponges. Our results provide invaluable basic data on A. mellifera brain circRNA patterns and a molecular basis for further study of the biological function of circRNAs in the development and immune response of honey bees.

Global identification of circular RNAs in imatinib (IM) resistance of chronic myeloid leukemia (CML) by modulating signaling pathways of circ_0080145/miR-203/ABL1 and circ 0051886/miR-637/ABL1

Imatinib (IM), targeting of BCR-ABL1 tyrosine kinase, is currently one of the first-line choices in the treatment of chronic myeloid leukemia (CML). This study aims to explore the molecular mechanisms underlying IM resistance in CML treatment. 108 CML patients were recruited and grouped according to their sensitivity to IM as the responder group (N = 66) and the non-responder group (N = 42). Real-time quantitative PCR (RT-qPCR) was performed to evaluate the expression of candidate circular RNAs (circRNAs), microRNA (miRNAs) and messenger RNA (mRNAs). No significant difference was noted regarding demographic and clinicopathological characteristics between the responder group and the non-responder group. The expression of circ_0080145, circ_0051886 and ABL1 mRNA was significantly increased, while the expression of miR-203 and miR-637 was decreased in the non-responder group as compared with the responders. By using in-silicon analysis, it was predicted that circ_0080145 and circ_0051886 targeted miR-203 and miR-637 respectively, and ABL1 was found to be shared direct target gene of miR-203 and miR-637. Ectopic over-expression of circ_0080145 and circ_0051886 respectively reduced the expression of miR-203 and miR-637. The expression of ABL1 mRNA/protein was most upregulated in culture cells co-transfected with circ_0080145 and circ_0051886 as compared with those cells individually transfected. This study established the signaling pathways of circ_0080145/miR-203/ABL1 and circ 0051886/miR-637/ABL1. The deregulation of circ_0080145 and circ_0051886 is, at least partially, responsible for the development of IM chemoresistance in CML by regulating expression of ABL1 via modulating expression of miR-203 and miR-637.

Identification of Circular RNAs in the Anterior Pituitary in Rats Treated with GnRH.

Simple SummaryThe pituitary gland, an important endocrine organ, can secrete a variety of reproductive hormones under the action of hypothalamus-secreted gonadotropin-releasing hormone. Circular RNAs are a class of RNA molecules with stable covalently closed circular structures. In this study, we performed RNA sequencing of GnRH-treated rats to identify differentially expressed circRNAs in the anterior pituitary. The results revealed 1433 related circRNAs, 14 of which were differentially expressed. We predicted targeted relationships between the differentially expressed circRNAs and FSHb-LHb-associated miRNAs. In all, a total of 14 circRNAs were identified that may act on the secretion and regulation of reproductive hormones in GnRH-treated rats.The pituitary gland, an important endocrine organ, can secrete a variety of reproductive hormones under the action of hypothalamus-secreted gonadotropin-releasing hormone (GnRH) and plays important roles in animal reproduction. Circular RNAs (circRNAs) are a class of RNA molecules with stable covalently closed circular structures. CircRNAs are equipped with miRNA response elements (MREs), which can regulate the expression of target genes by competitively binding miRNAs. However, whether the expression levels of circRNAs in the pituitary gland change under the action of GnRH and whether such changes can further affect the secretion of reproductive hormones are still unclear. In this study, we performed RNA sequencing (RNA-Seq) of GnRH-treated rats to identify differentially expressed circRNAs. The results revealed 1433 related circRNAs, 14 of which were differentially expressed. In addition, we randomly selected five differentially expressed circRNAs and tested their relative expression levels by RT-qPCR, the results of which were consistent with the RNA sequencing results. Finally, we predicted targeted relationships between the differentially expressed circRNAs and FSHb-LHb-associated miRNAs. In all, a total of 14 circRNAs were identified that may act on the secretion and regulation of reproductive hormones in GnRH-treated rats. Our expression profiles of circRNAs in the anterior pituitaries of rats treated with GnRH can provide insights into the roles of circRNAs in mammalian development and reproduction.

Advances in the Identification of Circular RNAs and Research Into circRNAs in Human Diseases.

Circular RNAs (circRNAs) are a class of endogenous non-coding RNAs (ncRNAs) with a closed-loop structure that are mainly produced by variable processing of precursor mRNAs (pre-mRNAs). They are widely present in all eukaryotes and are very stable. Currently, circRNA studies have become a hotspot in RNA research. It has been reported that circRNAs constitute a significant proportion of transcript expression, and some are significantly more abundantly expressed than other transcripts. CircRNAs have regulatory roles in gene expression and critical biological functions in the development of organisms, such as acting as microRNA sponges or as endogenous RNAs and biomarkers. As such, they may have useful functions in the diagnosis and treatment of diseases. CircRNAs have been found to play an important role in the development of several diseases, including atherosclerosis, neurological disorders, diabetes, and cancer. In this paper, we review the status of circRNA research, describe circRNA-related databases and the identification of circRNAs, discuss the role of circRNAs in human diseases such as colon cancer, atherosclerosis, and gastric cancer, and identify remaining research questions related to circRNAs.

Identification of circular RNAs as novel biomarkers and potentially functional competing endogenous RNA network for myelodysplastic syndrome patients.

Circular RNAs (circRNAs) have been identified to exert vital biological functions and can be used as new biomarkers in a number of tumors. However, little is known about the functions of circRNAs in myelodysplastic syndrome (MDS). Here, we aimed to investigate circRNA expression profiles and to investigate the functional and clinical value of circRNAs in MDS. Differential expression of circRNAs between MDS and control subjects was analyzed using circRNA arrays, in which we identified 145 upregulated circRNAs and 224 downregulated circRNAs. Validated by real‐time quantitative PCR between 100 MDS patients and 20 controls, three upregulated (hsa_circRNA_100352, hsa_circRNA_104056, and hsa_circRNA_104634) and three downregulated (hsa_circRNA_103846, hsa_circRNA_102817, and hsa_circRNA_102526) circRNAs matched the arrays. The receiver operating characteristic curve analysis of these circRNAs showed that the area under the curve was 0.7266, 0.8676, 0.7349, 0.7091, 0.8806, and 0.7472, respectively. Kaplan‐Meier survival analysis showed that only hsa_circRNA_100352, hsa_circRNA_104056, and hsa_circRNA_102817 were significantly associated with overall survival. Furthermore, we generated a competing endogenous RNA network focused on hsa_circRNA_100352, hsa_circRNA_104056, and hsa_circRNA_102817. Analyses using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes showed that the three circRNAs were linked with some important cancer‐related functions and pathways.

The identification of circular RNAs from peripheral blood mononuclear cells in systemic lupus erythematosus

BackgroundThe diagnosis of systemic lupus erythematosus (SLE) is complicated. This study explores the expression of circular RNAs (circRNAs), which are closed non-coding RNAs in which the 5′ and 3′ ends are covalently linked and which work by sponging microRNAs. CircRNAs were extracted from peripheral blood mononuclear cells (PBMCs) of SLE patients to identify novel circRNA species that might be used for SLE diagnosis.MethodsMicroarray was applied to screening circRNAs changes in PBMCs obtained from SLE patients (n = 10) and healthy participants (n = 10), paired for age and sex. We then verified the selected circRNAs in PBMCs using quantitative reverse transcription-polymerase chain reaction amplification (qRT-PCR) in another cohort, including ten paired SLE patients and healthy participants. The correlation between the differential circRNAs and clinical pathology of SLE were analyzed.Results182 up-regulated and 563 significantly down-regulated circRNAs in PBMCs of patients with SLE were identified. Besides, the qRT-PCR results were consistent with the microarray results. The correlation analysis revealed that has_circRNA_100236, has_circRNA_102489, and has_circRNA_101413 were correlated with positive anti-dsDNA, thrombocytopenia, and positive IgG, respectively. Lastly, their miRNAs targets and the binding sites were predicted.ConclusionWe identified some dysregulated circRNAs in PBMCs from SLE patients, and these circRNAs may be developed as the novel biomarkers for the diagnosis of SLE.

Identification of Circular RNAs in Hypothalamus of Gilts during the Onset of Puberty.

The disorders of puberty have shown negative outcomes on health of mammals, and the hypothalamus is thought to be the main regulator of puberty by releasing GnRH. Many studies show that the circular RNAs (circRNAs) might be implicated in the timing of puberty in mammals. However, the circRNAs in the hypothalamus of gilts have not been explored. To profile the changes and biological functions of circRNAs in the hypothalamus during the onset of puberty, RNA-seq was utilized to establish pre-, in-, and post-pubertal hypothalamic circRNAs profiles. In this study, the functions of hypothalamic circRNAs were enriched in the signaling pathway of neurotrophin, progesterone-mediated oocyte maturation, oocyte meiosis, insulin, ErbB, and mTOR, which have been highly suggested to be involved in the timing of puberty. Furthermore, 53 circRNAs were identified to be putative hypothalamus-specific expressed circRNAs, and some of them were exclusively expressed in the one of three pubertal stages. Moreover, 22 differentially expressed circRNAs were identified and chosen to construct the circRNA-miRNA-gene network. Moreover, 10 circRNAs were found to be driven by six puberty-related genes (ESR1, NF1, APP, ENPP2, ARNT, and DICER1). Subsequently, the expression changes of several circRNAs were confirmed by RT-qPCR. Collectively, the preliminary results of hypothalamic circRNAs provided useful information for the investigation of the molecular mechanism for the timing of puberty in gilts.

Identification of Circular RNAs by Multiple Displacement Amplification and Their Involvement in Plant Development.

With the innovative knowledge and bioinformatics tools in the identification and characterization of noncoding RNAs, circular RNA (circRNA) is added as a new member to the noncoding RNAs family. CircRNA enrichment by rRNA depletion/RNase R or poly-A removal/RNase R treatment followed by NGS analysis is the most frequently adopted method for circular RNA identification and characterization. In this chapter, we describe the multiple displacement amplification (MDA) as a convenient method to augment the identification of even the abysmally expressed circular RNAs at low sequencing depth. Total RNA, extracted at three different developmental stages of rice, is subjected to RiboMinus and RNase R treatment to deplete the linear RNAs. The enriched circular RNAs are reverse transcribed with random hexamers. The resulting cDNA is subjected to phi29 DNA polymerase amplification using exo-resistant random pentamers to yield high molecular weight dsDNA product, followed by Illumina sequencing at ten million paired end reads per sample. The sequence analysis yielded a promising number of circRNAs with the appreciable inclusion of differentially regulated and minimally expressed circRNAs at a comparatively reduced cost.