The recent discovery of circular RNAs (circRNAs) and characterization of their functional roles have opened a new avenue for understanding the biology of genomes. circRNAs have been implicated to play important roles in a variety of biological processes, but their precise functions remain largely elusive. Currently, a few approaches are available for novel circRNA prediction, but almost all these methods are intended for animal genomes. Considering that the major differences between the organization of plant and mammal genomes cannot be neglected, a plant-specific method is needed to enhance the validity of plant circRNA identification. In this study, we present CircPlant, an integrated tool for the exploration of plant circRNAs, potentially acting as competing endogenous RNAs (ceRNAs), and their potential functions. With the incorporation of several unique plant-specific criteria, CircPlant can accurately detect plant circRNAs from high-throughput RNA-seq data. Based on comparison tests on simulated and real RNA-seq datasets from Arabidopsis thaliana and Oryza sativa, we show that CircPlant outperforms all evaluated competing tools in both accuracy and efficiency. CircPlant is freely available at http://bis.zju.edu.cn/circplant.
Arabidopsis/metabolism* ; Oryza/metabolism* ; RNA, Circular/metabolism* ; RNA, Plant/metabolism* ; Sequence Analysis, RNA/methods*

Parasite-derived non-coding RNAs (ncRNAs) not only contribute to life activities of parasites, and microRNA (miRNA), long non-coding RNA (lncRNA), and circular RNA (circRNA) may generate a competitive endogenous RNA (ceRNA) regulatory network with host miRNAs and mRNAs via extracellular vesicles, thereby participating in infection and pathogenic processes. This article presents an overview of characterizing ncRNAs derived from parasites and the cross-species regulatory role of parasite-derived ncRNAs in host gene expression and its underlying mechanisms.
Animals ; Parasites ; Gene Regulatory Networks ; MicroRNAs/metabolism* ; RNA, Messenger/genetics* ; RNA, Circular/genetics* ; RNA, Competitive Endogenous

Circular RNAs (circRNAs) are a new class of non-coding RNAs, which have been confirmed to regulate insect gene expression and immune response through multiple manners such as competing endogenous RNA (ceRNA) regulatory network. Currently, function of circRNA in honey bee immune response remains unclear. In this study, PCR and Sanger sequencing were performed to validate the back splicing (BS) site of ame_circ_000115 (in short ac115). RT-qPCR was used to detect the expression profile of ac115 in larval guts of Apis mellifera ligustica stressed by Ascosphaera apis. Dual-luciferase reporter gene assay was conducted to verify the binding relationship between ac115 and ame-miR-13b. Interference of ac115 in larval guts was carried out by feeding specific siRNA, followed by determination of the effect of ac115 interference on expression of six genes relevant to host immune response. The results confirmed the existence of BS site within ac115. Compared with the un-inoculated group, the expression of ac115 in 4-day-old larval gut of the A. apis-inoculated group was up-regulated with extreme significance (P < 0.000 1), while that in 5- and 6-day-old larval guts were significantly up-regulated (P < 0.05). The brightness of specific band for ac115 in 4-, 5- and 6-day-old larval guts of the siRNA-circ_000115-fed group gradually became weak, whereas that of the siRNA-scrambl-fed group was pretty high without obvious variation. Compared with that of the siRNA-scramble-fed group, the expression of ac115 in 4-day-old larval gut of the siRNA-circ_000115-fed group was significantly down-regulated (P < 0.05), whereas that of the 5- and 6-day-old larval guts were down-regulated with extreme significance (P < 0.001). Ame-miR-13b was truly existed and expressed in A. m. ligustica larval guts, and there was true binding relationship between ac115 and ame-miR-13b. Compared with that of the siRNA-scramble-fed group, the expression of antimicrobial peptide genes hymenoptaecin and abaecin in 6-day-old larval gut of the siRNA-circ_000115-fed group was significantly up-regulated (P < 0.05), while that of ecdysone receptor (Ecr) was down-regulated with extreme significance (P < 0.01). These results indicate that ac115 is truly expressed in A. m. ligustica larval guts, BS site truly exists within ac115, and effective interference of ac115 in A. m. ligustica larval guts can be achieved via feeding siRNA. Moreover, ac115 potentially regulates Ecr expression through adsorption of ame-miR-13b and expression of hymenoptaecin and abaecin using a non-ceRNA manner, further participating in host stress-response.
Bees/genetics* ; Animals ; Larva/metabolism* ; RNA, Circular/genetics* ; RNA, Small Interfering/genetics* ; MicroRNAs/genetics*

Acute kidney injury (AKI) is a common clinical syndrome and an independent risk factor of chronic kidney disease and end-stage renal failure. At present, the treatments of AKI are still very limited and the morbidity and mortality of AKI are rising. Non-coding RNAs (ncRNAs), including microRNAs, long non-coding RNAs and circular RNAs (circRNAs), are RNAs that are transcribed from the genome, but not translated into proteins. It has been widely reported that ncRNA is involved in AKI caused by ischemia reperfusion injury (IRI), drugs and sepsis through different molecular biological mechanisms, such as apoptosis and oxidative stress response. Therefore, ncRNAs are expected to become a new target for clinical prevention and treatment of AKI and a new biomarker for early warning of the occurrence and prognosis of AKI. Here, the role and mechanism of ncRNA in AKI and the research progress of ncRNA as biomarkers are reviewed.
Acute Kidney Injury/metabolism* ; Humans ; MicroRNAs/metabolism* ; RNA, Circular ; RNA, Long Noncoding/genetics* ; RNA, Untranslated/genetics* ; Reperfusion Injury/genetics*

Objective: To identify the expression profile of circular RNA (circRNA) in placenta of pre-eclampsia (PE) pregnant women by high-throughput sequencing, and to construct the circRNA-microRNA (miRNA)-messenger RNA (mRNA) interaction network, so as to reveal the related pathways and regulatory mechanisms of PE. Methods: The clinical data and placentas of 42 women with PE (PE group) and 30 normal pregnant women (control group) who delivered in West China Second University Hospital from November 2019 to June 2021 were collected. (1) High-throughput sequencing was used to establish the differentially expressed circRNA profiles in placental tissues of 5 pairs of PE group and the control group. (2) Real-time quantitative PCR (qRT-PCR) was used to verify the expression levels of 6 differentially expressed circRNAs in placental tissues of PE group and control group. (3) Bioinformatics analysis was used to predict the target miRNA and analyze the co-expressed mRNA to construct a competitive endogenous RNA (ceRNA) network. The differentially expressed circRNAs were analyzed by Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. (4) Logistic regression analysis, Pearson correlation and Kendall's tau-b correlation analysis were used to test the correlation between the three differentially expressed circRNAs and the risk of PE and clinical characteristics. (5) circRNA_05393 was selected for subsequent functional study. Small interfering RNA (siRNA) and overexpression plasmid were used to knock down or increase the expression level of circRNA_05393 in trophoblast cell line HTR-8/SVneo cells, respectively. Transwell assay was used to detect the migration and invasion ability of the trophoblasts in vitro. Cell counting kit-8 assay was used to detect the proliferation ability of the trophoblasts. Results: (1) Seventy-two differentially expressed circRNAs were identified by high-throughput sequencing, of which 35 were up-regulated and 37 were down-regulated. (2) qRT-PCR showed that compared with the control group, circRNA_00673 (1.306±0.168 vs 2.059±0.242; t=2.356, P=0.021) and circRNA_07796 (1.275±0.232 vs 1.954±0.230; t=2.018, P=0.047) were significantly increased, while circRNA_05393 (1.846±0.377 vs 0.790±0.094; t=3.138, P=0.002) was significantly decreased. (3) The circRNA-miRNA-mRNA interaction network contained 3 circRNAs, 8 miRNAs and 53 mRNAs. GO functional annotation analysis showed that the biological process was mainly enriched in iron ion homeostasis, membrane depolarization during action potential and neuronal action potential. In terms of cellular components, they were mainly enriched in cytoskeleton and membrane components. In terms of molecular function, they were mainly enriched in the activity of voltage-gated sodium channel and basic amino acid transmembrane transporter. KEGG pathway enrichment analysis showed that mRNAs in the interaction network were mainly enriched in complement and coagulation cascade, glycine, serine and threonine metabolism, p53 signaling pathway and peroxisome proliferators-activated receptors (PPAR) signaling pathway. (4) Logistic regression analysis showed that down-regulation of circRNA_05393 expression was a risk factor for PE (OR=0.044, 95%CI: 0.003-0.596; P=0.019). Correlation analysis showed that circRNA_05393 was significantly correlated with systolic blood pressure and diastolic blood pressure in PE pregnant women (both P<0.05). (5) Knock down or overexpression of circRNA_05393 significantly reduced or increased the migration and invasion abilities of HTR-8/SVneo cells (all P<0.05), but had no significant effect on the ability of tube formation and proliferation (all P>0.05). Conclusions: The construction of circRNA expression profile in placenta and the exploration of circRNA-miRNA-mRNA interaction network provide the possibility to reveal the regulatory mechanism of specific circRNA involved in PE. Inhibition of circRNA_05393 may induce the progression of PE by reducing the migration and invasion of trophoblasts.
Female ; Humans ; Pregnancy ; MicroRNAs/metabolism* ; RNA, Circular/metabolism* ; RNA, Messenger/metabolism* ; Pre-Eclampsia/metabolism* ; Placenta/metabolism* ; RNA/metabolism* ; RNA, Small Interfering ; Gene Expression Profiling

Circular RNAs (circRNAs) are highly conserved and ubiquitously expressed noncoding RNAs that participate in multiple reproduction-related diseases. However, the expression pattern and potential functions of circRNAs in the testes of patients with non-obstructive azoospermia (NOA) remain elusive. In this study, according to a circRNA array, a total of 37 881 circRNAs were identified that were differentially expressed in the testes of NOA patients compared with normal controls, including 19 874 upregulated circRNAs and 18 007 downregulated circRNAs. Using quantitative real-time polymerase chain reaction (qRT-PCR) analysis, we confirmed that the change tendency of some specific circRNAs, including hsa_circ_0137890, hsa_circ_0136298, and hsa_circ_0007273, was consistent with the microarray data in another larger sample. The structures and characteristics of these circRNAs were confirmed by Sanger sequencing, and fluorescence in situ hybridization revealed that these circRNAs were primarily expressed in the cytoplasm. Bioinformatics analysis was used to construct the competing endogenous RNA (ceRNA) network, and numerous miRNAs that could be paired with circRNAs validated in this study were reported to be vital for spermatogenesis regulation. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses indicated that genes involved in axoneme assembly, microtubule-based processes, and cell proliferation were significantly enriched. Our data suggest that there are aberrantly expressed circRNA profiles in patients with NOA and that these circRNAs may help identify key diagnostic and therapeutic molecular biomarkers for NOA patients.
Male ; Humans ; RNA, Circular/genetics* ; Azoospermia/genetics* ; In Situ Hybridization, Fluorescence ; MicroRNAs/metabolism*

OBJECTIVE: Circular RNAs (circRNAs) participate in several important pathological processes and have been used in the diagnosis and treatment of various diseases. This study aimed to investigate the role of circRNAs in neural tube defects (NTDs). METHOD: We characterized circRNA-associated competitive endogenous RNA (ceRNA) networks in brain tissue of low folate -induced NTDs mouse at embryonic day 13.5 by high-throughput sequencing. The expression levels of Circzfp644, miR-20-5p and Gas7 were detected by RT-PCR. Gas7 and Circzfp644 functions were determined by miRNA-mimics and inhibitors in mouse teratocarcinoma cells (F9 cells), and luciferase gene reporter assay was assessed in the F9 cells. In addition, the expression levels of Circzfp644, miR-20-5p and Gas7 were determined by Nanostring in human NTDs tissues. RESULTS: We detected 57 circRNA transcripts, 16 miRNAs, and 148 mRNAs that were significantly dysregulated in NTDs brain tissues compared with their expression levels in control (normal) tissues. Circzfp644 shared miRNA response elements with the growth arrest specific 7 ( Gas7) gene and competitively bound with miR-20-5p to increase the expression of Gas7. Downregulation of Circzfp644 and Gas7 and upregulation of miR-20-5p were found in human NTD tissue. CONCLUSION This study provides new perspectives on the role of circRNAs in nervous system development and the pathogenesis of NTDs.
Humans ; Animals ; Mice ; RNA, Circular/genetics* ; MicroRNAs/metabolism* ; Down-Regulation ; Neural Tube Defects/genetics* ; Folic Acid

With the development of high-throughput sequencing technology, circular RNAs (circRNAs) have gradually become a hotspot in the research on non-coding RNA. CircRNAs are produced by the covalent circularization of a downstream 3' splice donor and an upstream 5' splice acceptor through backsplicing, and they are pervasive in eukaryotic cells. CircRNAs used to be considered byproducts of false splicing, whereas an explosion of related studies in recent years has disproved this misconception. Compared with the rich studies of circRNAs in animals, the study of circRNAs in plants is still in its infancy. In this review, we introduced the discovery of plant circRNAs, the discovery of plant circRNAs, the circularization feature, expression specificity, conservation, and stability of plant circRNAs and expounded the identification tools, main types, and biogenesis mechanisms of circRNAs. Furthermore, we summarized the potential roles of plant circRNAs as microRNA (miRNA) sponges and translation templates and in response to biotic/abiotic stress, and briefed the degradation and localization of plant circRNAs. Finally, we discussed the challenges and proposed the future directions in the research on plant circRNAs.
Animals ; MicroRNAs/metabolism* ; Organelle Biogenesis ; Plants/metabolism* ; Protein Biosynthesis/physiology* ; RNA, Circular/metabolism* ; RNA, Plant/metabolism* ; Research/trends* ; Stress, Physiological/genetics*

S1 nuclease (from Aspergillus oryzae) is a specific enzyme to degrade single stranded DNA or RNA molecules. It has been reported to be able to convert superhelical circular DNA molecules into open circle or linear forms under certain conditions, but this function has not been well explored. In order to use the action of S1 nuclease to linearize circular DNA and develop a novel way of cloning microcircular DNAs, the pUC19 was used to investigate the relationship between the linearization efficiency of S1 nuclease and the amount of enzyme used. By this way the optimal conditions for linearization of circular DNAs by S1 nuclease would be determined. 0.3u to 17u S1 nuclease per 100ng pUC19 DNA was added into a 25 microL system, respectively, to perform the reaction. The effectiveness of enzyme digestion was realized by electrophoresis in a 1.2% agarose gel. The results showed that along with the increase in enzyme amount from 0.3u to 17u a gradual decrease in the superhelical form, a gradual increase in the linear form and then in the circular form was obvious. The conversion from superhelical form to linear and circular form was directly related to the enzyme amount used. A higher proportion of linear DNA molecules was achieved by using 5 to 17u S1 nuclease per 100ng DNA. Besides, electrophoretic mobility of the S1 nuclease-linearized pUC19 was the same as that of the linear form produced by restriction enzyme digestion. According to the result of phiX174 digested by S1 nuclease it has been proposed that the enzyme cleaves first randomly on one site of one strand, thus converting the superhelical molecules into open circle form, and then on the same site of the complementary strand to produce the linear form. Therefore, the S1 nuclease-linearized DNA molecules are intact in the sense of their length and can be used for cloning. The plasmid-like DNA pC3 from cucumber mitochondria is a double stranded circular DNA molecule with about 550bp and the smallest known plasmid-like DNA in eukaryotic mitochondria. Many attempts have been made to linearize the molecule by using restriction enzymes but failed. Therefore, S1 nuclease was used to linearize pC3 based on the results obtained with pUC19. The linearized pC3 DNA molecules formed a very sharp band in a 2.5% agarose gel after electrophoresis. They were then recovered from the gel, added an "A" tail and ligated with T-vector. After transformation into E. coli JM109 cells, the positive clones were, screened by the blue-white selection. The insert was then cut using restriction enzymes EcoRI and Pst I. The result of electrophoresis shows that the electrophoretic mobility of the insert is just the same as that predicted. A 32 P-labled probe was synthesized using pC3 as the template and Southern blot analysis was carried out. The result shows that the inserted DNA is hybridized to the probe, which indicates that the cloned DNA fragment is from pC3. The sequence information of the insert shows that the plasmid-like DNA pC3 was 537bp in length. The nucleotide sequence was deposited in the GenBank (the accession number is AF522195).
Blotting, Southern ; Cloning, Molecular ; methods ; DNA, Circular ; genetics ; metabolism ; Fungal Proteins ; genetics ; metabolism ; Molecular Sequence Data ; Single-Strand Specific DNA and RNA Endonucleases ; genetics ; metabolism

To investigate the mechanism by which Cangxi Tongbi Capsules promote chondrocyte autophagy to inhibit knee osteoarthritis(KOA) progression by regulating the circRNA＿0008365/miR-1271/p38 mitogen-activated protein kinase(MAPK) pathway. The cell and animal models of KOA were established and intervened with Cangxi Tongbi Capsules, si-circRNA＿0008365, si-NC, and Cangxi Tongbi Capsules combined with si-circRNA＿0008365. Flow cytometry and transmission electron microscopy were employed to determine the level of apoptosis and observe autophagosomes, respectively. Western blot was employed to reveal the changes in the protein levels of microtubule-associated protein light chain 3(LC3)Ⅱ/Ⅰ, Beclin-1, selective autophagy junction protein p62/sequestosome 1, collagen Ⅱ, a disintegrin and metalloproteinase with thrombospondin motifs 5(ADAMTS-5), and p38 MAPK. The mRNA levels of circRNA＿0008365, miR-1271, collagen Ⅱ, and ADAMTS-5 were determined by qRT-PCR. Hematoxylin-eosin staining was employed to reveal the pathological changes of the cartilage tissue of the knee, and enzyme-linked immunosorbent assay to measure the levels of interleukin-1β(IL-1β) and tumor necrosis factor-alpha(TNF-α). The chondrocytes treated with IL-1β showed down-regulated expression of circRNA＿0008365, up-regulated expression of miR-1271 and p38 MAPK, lowered autophagy level, increased apoptosis rate, and accelerated catabolism of extracellular matrix. The intervention with Cangxi Tongbi Capsules up-regulated the expression of circRNA＿0008365, down-regulated the expression of miR-1271 and p38 MAPK, increased the autophagy level, decreased the apoptosis rate, and weakened the catabolism of extracellular matrix. However, the effect of Cangxi Tongbi Capsules was suppressed after interfering with circRNA＿0008365. The in vivo experiments showed that Cangxi Tongbi Capsules dose-dependently inhibited the p38 MAPK pathway, enhanced chondrocyte autophagy, and mitigated articular cartilage damage and inflammatory response, thereby inhibiting the progression of KOA in rats. This study indicated that Cangxi Tongbi Capsules promoted chondrocyte autophagy by regulating the circRNA＿0008365/miR-1271/p38 MAPK pathway to inhibit the development of KOA.
Rats ; Animals ; Chondrocytes ; Osteoarthritis, Knee/pathology* ; RNA, Circular/pharmacology* ; p38 Mitogen-Activated Protein Kinases/metabolism* ; MicroRNAs/metabolism* ; Apoptosis ; Autophagy/genetics* ; Collagen/metabolism*