circRNA Transcripts Research Articles

Evaluation of circANKLE2 & circL3MBTL4 -RNAs Expression in Fertile and Infertile Men.

There are many factors that affect male fertility such as chronic health problems, psychological factors, and illnesses. Male infertility can be caused abnormal sperm function, low sperm production or even blockages that prevent the delivery of sperm. The aim of the work is to determine the expression pattern of the circularANKLE2 and circularL3MBTL4 RNA in spermatozoa from fertile and infertile males, as well as the relationship between these circRNA transcripts and sperm quality. The study involved two groups: a control group comprising 40 healthy, fertile men and an experimental group of 90 infertile males. Semen samples were collected and processed for analysis using computer-assisted semen analysis. Following RNA extraction from sperm samples, reverse transcription and real-time PCR were performed to assess the levels of circular ANKLE2 and circular L3MBTL4 RNA. There was a significant up-regulation of circularANKLE2 RNA expression (p < 0.05), and a significant down-regulation of circularL3MBTL4 RNA expression (p < 0.05) in asthenozoospermia, astheno-teratozoospermia, and oligo-astheno-teratozoospermia groups, as well as, in immature spermatozoa separated from normozoospermic samples. Moreover, the altered expression of both circular L3MBTL4 and circular ANKLE2 RNA showed significant correlations with the associated sperm parameters. In conclusion, the expression of circular ANKLE2 RNA and circular L3MBTL4 RNA may play a significant role in male fertility and could serve as potential biomarkers of sperm quality, warranting further investigation for their application in infertility diagnostics.

Investigating Full-Length circRNA Transcripts to Reveal circRNAMediated Regulation of Competing Endogenous RNAs in Gastric Cancer

Background: Circular RNAs (circRNAs) play important regulatory roles in the progression of gastric cancer (GC), but the exact mechanisms governing their regulation remain incompletely understood. Prior studies typically used back-spliced junctions (BSJs) to represent a range of circRNA isoforms, overlooking the prevalence of alternative splicing (AS) events within circRNAs, which could lead to unreliable or even incorrect conclusions in subsequent analyses, hindering our comprehension of the specific functions of circRNAs in GC. Objective: This study aimed to explore the potential functional roles of the dysregulated circRNA transcripts in GC and provide new biomarkers and effective novel therapeutic strategies for GC treatment. Methods: RNA-seq data with rRNA depletion and RNase R treatment was employed to characterize the expression profiles of circRNAs in GC, and RNA-seq data only with rRNA depletion was employed to identify differentially expressed mRNAs in GC. Based on the full-sequence information and accurate isoform-level quantification of circRNA transcripts calculated by the CircAST tool, we performed a series of bioinformatic analyses. A circRNA-miRNA-hub gene regulatory network was constructed to reveal the circRNA-mediated regulation of competing endogenous RNAs in GC, and then the protein-protein interaction (PPI) network was built to identify hub genes. Results: A total of 18,398 circular transcripts were successfully reconstructed in the samples. Herein, 351 upregulated and 177 downregulated circRNA transcripts were identified. Functional enrichment analysis revealed that their parental genes were strongly associated with GC. After several screening steps, 19 dysregulated circRNA transcripts, 40 related miRNAs, and 65 target genes (mRNAs) were selected to construct the ceRNA network. Through PPI analysis, five hub genes (COL5A2, PDGFRB, SPARC, COL1A2, and COL4A1) were excavated. All these hub genes may play vital roles in gastric cancer cell proliferation and invasion. Conclusion: Our study revealed a comprehensive profile of full-length circRNA transcripts in GC, which could provide potential prognostic biomarkers and targets for GC treatment. The results would be helpful for further studies on the biological roles of circRNAs in GC and offer new mechanistic insights into the pathogenesis of GC.

Decoding protein binding landscape on circular RNAs with base-resolution transformer models

Circular RNAs (circRNAs), a class of endogenous RNA with a covalent loop structure, can regulate gene expression by serving as sponges for microRNAs and RNA-binding proteins (RBPs). To date, most computational methods for predicting RBP binding sites on circRNAs focus on circRNA fragments instead of circRNAs. These methods detect whether a circRNA fragment contains binding sites, but cannot determine where are the binding sites and how many binding sites are on the circRNA transcript. We report a hybrid deep learning-based tool, CircSite, to predict RBP binding sites at single-nucleotide resolution and detect key contributed nucleotides on circRNA transcripts. CircSite takes advantage of convolutional neural networks (CNNs) and Transformer for learning local and global representations of circRNAs binding to RBPs, respectively. We construct 37 datasets of circRNAs interacting with proteins for benchmarking and the experimental results show that CircSite offers accurate predictions of RBP binding nucleotides and detects key subsequences aligning well with known binding motifs. CircSite is an easy-to-use online webserver for predicting RBP binding sites on circRNA transcripts and freely available at http://www.csbio.sjtu.edu.cn/bioinf/CircSite/.

Genome-wide characteristics and potential functions of circular RNAs from the embryo muscle development in Chengkou mountain chicken.

The Chengkou mountain chicken, a native Chinese poultry breed, holds significant importance in the country's poultry sector due to its delectable meat and robust stress tolerance. Muscle growth and development are pivotal characteristics in poultry breeding, with muscle fiber development during the embryonic period crucial for determining inherent muscle growth potential. Extensive evidence indicates that non-coding RNAs (ncRNAs) play a regulatory role in muscle growth and development. Among ncRNAs, circular RNAs (circRNAs), characterized by a closed-loop structure, have been shown to modulate biological processes through the regulation of microRNAs (miRNAs). This study seeks to identify and characterize the spatiotemporal-specific expression of circRNAs during embryonic muscle development in Chengkou mountain chicken, and to construct the potential regulatory network of circRNAs-miRNA-mRNAs. The muscle fibers of HE-stained sections became more distinct, and their boundaries were more defined over time. Subsequent RNA sequencing of 12 samples from four periods generated 9,904 novel circRNAs, including 917 differentially expressed circRNAs. The weighted gene co-expression network analysis (WGCNA)-identified circRNA source genes significantly enriched pathways related to cell fraction, cell growth, and muscle fiber growth regulation. Furthermore, a competitive endogenous RNA (ceRNA) network constructed using combined data of present and previous differentially expressed circRNAs, miRNA, and mRNA revealed that several circRNA transcripts regulate MYH1D, MYH1B, CAPZA1, and PERM1 proteins. These findings provide insight into the potential pathways and mechanisms through which circRNAs regulate embryonic muscle development in poultry, a theoretical support for trait improvement in domestic chickens.

Systematic analysis of various RNA transcripts and construction of biological regulatory networks at the post-transcriptional level for chronic obstructive pulmonary disease

BackgroundAlthough chronic inflammation, oxidative stress, airway remodeling, and protease-antiprotease imbalance have been implicated in chronic obstructive pulmonary disease (COPD), the exact pathogenesis is still obscure. Gene transcription and post-transcriptional regulation have been taken into account as key regulators of COPD occurrence and development. Identifying the hub genes and constructing biological regulatory networks at the post-transcriptional level will help extend current knowledge on COPD pathogenesis and develop potential drugs.MethodsAll lung tissues from non-smokers (n = 6), smokers without COPD (smokers, n = 7), and smokers with COPD (COPD, n = 7) were collected to detect messenger RNA (mRNA), microRNA (miRNA), circular RNA (circRNA), and long non-coding RNA (lncRNA) expression and identify the hub genes. Biological regulatory networks were constructed at the post-transcriptional level, including the RNA-binding protein (RBP)-hub gene interaction network and the competitive endogenous RNA (ceRNA) network. In addition, we assessed the composition and abundance of immune cells in COPD lung tissue and predicted potential therapeutic drugs for COPD. Finally, the hub genes were confirmed at both the RNA and protein levels.ResultsAmong the 20 participants, a total of 121169 mRNA transcripts, 1871 miRNA transcripts, 4244 circRNA transcripts, and 122130 lncRNA transcripts were detected. There were differences in the expression of 1561 mRNAs, 48 miRNAs, 33 circRNAs, and 545 lncRNAs between smokers and non-smokers, as well as 1289 mRNAs, 69 miRNAs, 32 circRNAs, and 433 lncRNAs between smokers and COPD patients. 18 hub genes were identified in COPD. TGF-β signaling and Wnt/β-catenin signaling may be involved in the development of COPD. Furthermore, the circRNA/lncRNA-miRNA-mRNA ceRNA networks and the RBP-hub gene interaction network were also constructed. Analysis of the immune cell infiltration level revealed that M2 macrophages and activated NK cells were increased in COPD lung tissues. Finally, we identified that the ITK inhibitor and oxybutynin chloride may be effective in treating COPD.ConclusionsWe identified several novel hub genes involved in COPD pathogenesis. TGF-β signaling and Wnt/β-catenin signaling were the most dysregulated pathways in COPD patients. Our study constructed post-transcriptional biological regulatory networks and predicted small-molecule drugs for the treatment of COPD, which enhanced the existing understanding of COPD pathogenesis and suggested an innovative direction for the therapeutic intervention of the disease.

Characterization of CircRNA-Associated CeRNA Networks in Folate Deficiency-Induced Neural Tube Defects

ObjectiveCircular 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). MethodWe 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. ResultsWe 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. ConclusionThis study provides new perspectives on the role of circRNAs in nervous system development and the pathogenesis of NTDs.

Hsa_circ_0023826 protects against glaucoma by regulating miR-188-3p/MDM4 axis.

Circular RNAs (circRNAs) are characterized as a class of covalently closed circRNA transcripts and are associated with various cellular processes and neurological diseases by sponging microRNAs. The most common feature of glaucoma, a form of retinal neuropathy, is the loss of retinal ganglion cells. Although the pathogenesis of glaucoma is not fully understood, elevated intraocular pressure is undoubtedly the only proven modifiable factor in the classic glaucoma model. This study investigated the role of circ_0023826 in glaucoma-induced retinal neurodegeneration by modifying the miR-188-3p/mouse double minute 4 (MDM4) axis. The expression pattern of circ_0023826 was analyzed during retinal neurodegeneration. The effect of circ_0023826, miR-188-3p, and MDM4 on retinal neurodegeneration in vivo was assessed by visual behavioral testing and HandE staining in glaucoma rats, while that on in vitro retinal ganglion cells (RGCs) was evaluated by MTT assay, flow cytometry, Western blot, and ELISA. Bioinformatics analysis, RNA pull-down assay, luciferase reporter assay were performed to reveal the regulatory mechanism of circ_0023826-mediated retinal neurodegeneration. Circ_0023826 expression was downregulated during retinal neurodegeneration. Upregulating circ_0023826 attenuated the visual impairment in rats and promoted the survival of RGCs in vitro. Circ_0023826 acted as a sponge of miR-188-3p sponge, resulting in increased expression of MDM4. MDM4 silencing or miR-188-3p upregulation reversed the protective effect of upregulated circ_0023826 on glaucoma-induced neuroretinal degeneration in vitro and in vivo. Overall, circ_0023826 protects against glaucoma by regulating the miR-188-3p/MDM4 axis, and targeted intervention of circ_0023826 expression is a promising therapeutic strategy for the treatment of retinal neurodegeneration.

CircRNA Galntl6 sponges miR-335 to ameliorate stress-induced hypertension through upregulating Lig3 in rostral ventrolateral medulla

Rostral ventrolateral medulla (RVLM) is thought to serve as a major vasomotor center that participates in controlling the progression of stress-induced hypertension (SIH). Circular RNAs (circRNAs) perform important functions in the regulation of diverse physiological and pathological processes. However, information concerning the functions of RVLM circRNAs on SIH remains limited. RNA sequencing was performed to profile circRNA expression in RVLMs from SIH rats, which were induced by electric foot shocks and noises. The functions of circRNA Galntl6 in reducing blood pressure (BP) and its potential molecular mechanisms on SIH were investigated via various experiments, such as Western blot and intra-RVLM microinjection. A total of 12,242 circRNA transcripts were identified, among which circRNA Galntl6 was dramatically downregulated in SIH rats. The upregulation of circRNA Galntl6 in RVLM effectively decreased the BP, sympathetic outflow, and neuronal excitability in SIH rats. Mechanistically, circRNA Galntl6 directly sponged microRNA-335 (miR-335) and restrained it to reduce oxidative stress. Reintroduction of miR-335 observably reversed the circRNA Galntl6-induced attenuation of oxidative stress. Furthermore, Lig3 can be a direct target of miR-335. MiR-335 inhibition substantially increased the expression of Lig3 and suppressed oxidative stress, and these favorable effects were blocked by Lig3 knockdown. CircRNA Galntl6 is a novel factor that impedes SIH development, and the circRNA Galntl6/miR-335/Lig3 axis represents one of the possible mechanisms. These findings demonstrated circRNA Galntl6 as a possibly useful target for the prevention of SIH.

Genetic regulation of circular RNA expression in human aortic smooth muscle cells and vascular traits.

Circular RNAs (circRNAs) are a class of non-coding RNAs that have cell-type-specific expression and are relevant in cardiovascular disease. Aortic smooth muscle cells (SMCs) play a crucial role in cardiovascular disease. In this study, we employed a systems genetics approach to identify SMC circRNA transcripts and their relevance in cardiovascular traits across the genome. We quantified circRNA expression across 151 quiescent and proliferative human aortic SMCs from donors of various genetic ancestries. We identified 1,589 expressed circRNAs. Between quiescent and proliferative SMCs, we identified 173 differentially expressed circRNAs. To characterize the genetic regulation of circRNA expression, we associated the genotypes of 6.3 million single nucleotide polymorphisms (SNPs) with circRNA abundance and found 96 circRNAs that were associated with genetic loci. Three SNPs were associated with circRNA expression in proliferative SMCs but not quiescent SMCs. We identified six SNPs that had distinct association directions with circRNA isoforms from the same gene. Lastly, to identify the relevance of circRNAs in cardiovascular disease, we overlapped genetic loci associated with circRNA expression with vascular disease-related genome-wide association studies loci. We identified 14 blood pressure, one myocardial infarction, and three coronary artery disease loci, which were associated with a circRNA transcript but not an mRNA transcript. Overall, our results provide insight into the genetic basis of vascular disease traits mediated by circRNA expression.

CircRNA screening and ceRNA network construction for milk fat metabolism in dairy cows.

Milk fat is one of the main reference elements for evaluating milk quality and is a primary objective trait in dairy cattle breeding. In recent years, circular RNAs (circRNAs) have been found to play crucial roles in many biological processes. However, the function and expression profiles of circRNAs in milk fat synthesis in cows are not completely understood. We performed RNA sequencing to analyze the genome-wide expression of circRNA transcripts in bovine mammary epithelial cells (BMECs) from cows with extreme differences in milk fat percentage. We identified candidate differential circRNAs associated with milk fat metabolism using functional enrichment analysis and constructed a lipid metabolism-related competing endogenous RNA (ceRNA) interactive regulatory network. A total of 290 circRNAs were significantly differentially expressed (DE-circRNAs) in high milk fat percentage (HMF) cows compared to that in low milk fat percentage (LMF) cows. Of the 290 circRNAs, 142 were significantly upregulated and 148 were significantly downregulated. Enrichment analysis (Gene Ontology and Kyoto Encyclopedia of Genes and Genomes) identified four DE-circRNAs (circ_0001122, circ_0007367, circ_0018269, and circ_0015179) that potentially regulate milk fat metabolism. Among them, circ_0001122, circ_0007367, and circ_0015179 had relatively high expression levels in cow mammary gland tissue compared to other tissues (heart, liver, kidney, uterus, ovaries, and small intestine) of cows. The regulatory networks circ_0001122:miR-12043:LIPG, circ_0007367:miR-331-3p:CIDEA/PML, and circ_0018269:miR-11989:RORC/HPX are potential networks to explore the mechanism of milk fat regulation. These results reveal the possible role of circRNAs in milk fat metabolism in dairy cows. Several important circRNAs and ceRNAs affecting milk fat synthesis were identified, providing insights into the complex biology of milk fat synthesis as well as a novel theoretical perspective for future research on lactation, milk quality, and breed improvement in dairy cows.

Identification and Functional Prediction of Circular RNAs Related to Growth Traits and Skeletal Muscle Development in Duroc pigs.

Porcine skeletal muscle is a highly heterogeneous tissue type, and the Longissimus Dorsi muscle (LDM), as the most economical and physiologically metabolized skeletal muscle in pigs, has always been the focus of research and improvement in pig molecular breeding. Circular RNA, as an important new member of regulatory non-coding RNA after microRNA, has become a frontier hot spot in life science research. This study aims to explore candidate circRNAs related to growth, meat quality, and skeletal muscle development among Duroc pigs with different average daily gain (ADG). Eight pigs were selected and divided into two groups: H group (high-ADG) and L group (low-ADG), followed by RNA-Seq analysis to identify circRNAs. The results showed that backfat at 6-7 rib (BF) and Intramuscular fat (IMF) content in the H group was significantly lower than L group, but ribeye area (REA) in the H group was higher than in the L group. In RNA-seq, 296 Differentially expressed (DE) circRNAs (157 upregulated and 139 downregulated) were identified and exons flanking long introns are easier to circularize to produce circRNAs. Most of the DE circRNAs were enriched in Quantitative trait locus (QTL) regions related to meat quality and growth traits. In addition, a gene can produce one or more circRNA transcripts. It was also found that the source genes of DE circRNAs were enriched in MAPK, FoXO, mTOR, PI3K-Akt, and Wnt signaling pathways. The results showed that different ADG, carcass, and meat quality traits among half-sibling Duroc pigs with the same diet may be due to the DE circRNAs related to skeletal muscle growth and development.

CircFL-seq, A Full-length circRNA Sequencing Method.

Due to overlapping sequences with linear cognates, identifying internal sequences of circular RNA (circRNA) remains a challenge. Recently, we have developed a full-length circRNA sequencing method (circFL-seq) and computational pipeline, to profile ordinary and fusion circRNA at the isoform level. Compared to short-read RNA-seq, rolling circular reverse transcription and nanopore long-read sequencing of circFL-seq make circRNA reads more than tenfold enriched, and show advantages for identification of both short (<100 nt) and long (>2,000 nt) circRNA transcripts. circFL-seq allows identification of differential alternative splicing suggested wide application prospects for functional studies of internal sequences in circRNAs. In addition, the experimental protocol and computational pipeline of circFL-seq shows better sensitivity and precision for identification of back-splicing junctions than current long-read sequencing methods. Together, the accurate identification and quantification of full-length circRNAs makes circFL-seq a potential tool for large-scale screening of functional circRNAs.

Hsa_circ_0001275 Is One of a Number of circRNAs Dysregulated in Enzalutamide Resistant Prostate Cancer and Confers Enzalutamide Resistance In Vitro

Simple SummaryAlthough newer generations of androgen deprivation therapy such as enzalutamide are providing hope, it is clinically challenging to deliver effective therapy to individuals with metastatic castrate-resistant prostate cancer. Between 20–40% of patients have intrinsic resistance to therapy and all patients will ultimately experience disease progression due to acquired resistance, which is a significant clinical dilemma. The aim of our study was to evaluate the role of circular RNAs (circRNAs) in enzalutamide-resistant prostate cancer as part of the effort to identify useful biomarkers for patient selection and potential new therapeutic targets. We confirmed that hsa_circ_0001275 was highly upregulated in an enzalutamide resistant cell line and demonstrated that its overexpression resulted in increased enzalutamide resistance. Our data showed that hsa_circ_0001275 was not expressed abundantly in patient plasma samples, however, a trend of expression was evident which paralleled disease activity indicating a possible association with enzalutamide resistance. Overall, we have provided evidence that hsa_circ_0001275 promotes enzalutamide resistance and thus may serve as a potential therapeutic target.Background: Enzalutamide is part of the treatment regimen for metastatic castration-resistant prostate cancer (MCRPC). However, both intrinsic and acquired resistance to the drug remain substantial clinical quandaries. circRNAs, a novel type of non-coding RNA, have been identified in a number of cancers including prostate cancer and have been associated with cancer development and progression. circRNAs have shown great potential as clinically useful blood-based ‘liquid biopsies’ and as therapeutic targets in prostate cancer. The aim of this study was to examine the role of circRNA transcripts in enzalutamide-resistant prostate cancer cells and assess their utility as biomarkers. Methods: An isogenic cell line model of enzalutamide resistance was subjected to circRNA microarray profiling. Several differentially expressed circRNAs, along with their putative parental genes were validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). circRNAs of interest were stably overexpressed in the control cell line and drug sensitivity was assessed using an ELISA-based proliferation assay. The candidate circRNA, hsa_circ_0001275, was measured in patient plasma samples using RT-droplet digital PCR (RT-ddPCR). Results: hsa_circ_0001275 and its parental gene, PLCL2, were significantly up-regulated in strongly resistant clones vs. control (p < 0.05). Overexpression of hsa_circ_0001275 in the control cell line resulted in increased resistance to enzalutamide (p < 0.05). While RT-ddPCR analysis of hsa_circ_0001275 expression in plasma samples of 44 clinical trial participants showed a trend that mirrored the stages of disease activity (as defined by PSA level), the association did not reach statistical significance. Conclusions: Our data suggest that increased levels of hsa_circ_0001275 contribute to enzalutamide resistance. hsa_circ_0001275 plasma expression showed a trend that mirrors the PSA level at specific disease time points, indicating that circRNAs mirror disease recurrence and burden and may be associated with enzalutamide resistance.

Expression Profiles and Bioinformatics Analysis of Full-length circRNA Isoforms in Gliomas

Background: Circular RNAs (circRNAs) are a newly discovered type of non-coding RNA, which have been demonstrated to act as microRNA (miRNA) “sponges” to modulate gene expression. Emerging evidence has confirmed that circRNAs take part in many biological processes in a variety of malignant tumors, including gliomas, suggesting that they could serve as biomarkers or therapeutic targets for tumors. The purpose of this study was to explore the roles of circRNAs in gliomas and to provide valuable clues for clinical diagnosis and treatment. Methods: RNA-seq data with poly(A)-/RNase R treatment was employed to investigate the expression profiles of circRNAs in tumor and paracancerous tissues derived from glioma patients. CircAST was used for full-length circRNA reconstruction and quantification. Bioinformatics analyses, including GO enrichment and KEGG pathway analyses, were performed to reveal the potential biological process and pathways of their host genes. A circRNA-miRNA interaction network was constructed to depict the interactions of the dysregulated circRNA transcripts with miRNAs. Results: A total of 20,474 circular transcripts that originated from 16,022 circRNAs were successfully reconstructed in the samples. We detected 646 upregulated and 112 downregulated circular transcripts in tumor tissues compared with paracancerous tissues. GO analysis revealed that their host genes may be related to positive regulation of GTPase activity, regulation of synaptic transmission, and glutamatergic and dendrite morphogenesis in the cytoplasm and cytosol. KEGG pathway analysis showed that the glutamatergic synapse, neurotrophin signaling pathway, and ErbB signaling pathway may be linked to the occurrence and development of gliomas. Conclusion: Our study revealed a comprehensive profile of differentially expressed circRNA transcripts in gliomas, indicating that aberrantly expressed circRNAs might play important roles in the occurrence and development of human gliomas.

Profiling Analysis of Circular RNA and mRNA in Human Temporal Lobe Epilepsy with Hippocampal Sclerosis ILAE Type 1.

Hippocampal sclerosis (HS) is the most common surgical pathology associated with temporal lobe epilepsy (TLE). However, the cause of TLE with or without HS remains unknown. Our current study aimed to illustrate the essential molecular mechanism that is potentially involved in the pathogenesis of TLE-HS and to shed light on the transcriptional changes associated with hippocampal sclerosis. Compared to no-HS group, 341 mRNA transcripts and 131 circRNA transcripts were differentially expressed in ILAE type 1 group. The raw sequencing data have been deposited into sequence-read archive (SRA) database under accession number PRJNA699348.Gene Ontology analysis demonstrated that the dysregulated genes were associated with the biological processes of vesicle-mediated transport. Enrichment analysis demonstrated that dysregulated genes were involved mainly in the MAPK signal pathway. Subsequently, A total of 441 known or predicted interactions were formed among DEGs, and the most important module was detected in the PPI network using the MCODE plug-in. There were mainly four functional modules enriched: ER to Golgi transport vesicle membrane, Basal transcription factors, GABA-gated chloride ion channel activity, CENP-A containing nucleosome assembly. A circRNA-mRNA co-expression network was constructed including 5 circRNAs(hsa_circ_0025349, hsa_circ_0002405, hsa_circ_0004805, hsa_circ_0032254, and hsa_circ_0032875) and three mRNAs (FYN, SELENBP1, and GRIPAP1) based on the normalized mRNA signal intensities. This is the first to report the circRNAs and mRNAs expression profile of surgically resected hippocampal tissues from TLE patients of ILAE-1 and no-HS, and these results may provide new insight into the transcriptional changes associated with this pathology.

The Effect of Infertility on the Liver Structure, Endocrinology, and Gene Network in Japanese Flounder.

Simple SummaryInfertility is a serious disorder that is characterized by the absence of fecundity in an individual. Not only does infertility in human beings affect reproduction, it also impacts hormonal secretion, metabolism, and the immune system. The doubled haploid (DH) that is induced by artificial mitogynogenesis or androgenesis in fish often results in infertility. The causes of infertility and its effects on other organs in the DH Japanese flounder (Paralichthys olivaceus) have not yet been established. Due to many aspects, the reproductive biology of this species is largely unknown or incomplete. Here, we compared the liver structure and hormone levels between fertile and infertile Japanese flounders, constructed ceRNA networks, and performed the integrated analysis of transcriptomics and proteomics. We concluded that gonadal infertility is associated with not only changes in histological structure and hormone secretion, but also changes in metabolism, immunity, and signal transduction networks in the liver. We also identified and characterized the differently expressed non-protein coding transcripts that are involved in liver functions in response to infertility. This study expanded our understanding of the molecular mechanisms of infertility on fish.The liver can synthesize vitellogenin, the precursor of vitellin, which is needed for oocyte development and maturation. Here, we investigated the effects of infertility on liver structure, hormone regulation, and gene and protein networks in Japanese flounder (Paralichthys olivaceus). Results showed that the liver of infertile fish had fewer vacuoles and significantly lower serum vitellogenin (VTG) level than in liver of fertile fish. Whole transcriptomics analysis between infertile and fertile groups identified 2076 significantly differentially expressed (DE) mRNAs, 431 DE lncRNAs, 265 DE circRNAs, and 53 DE miRNAs. Proteomics analysis identified 838 DE proteins. Integrated analysis of whole transcriptomics and proteomics revealed 60 significantly DE genes and proteins associated with metabolism, immunity, signal transduction, and steroid biosynthesis. Moreover, non-coding RNA (miRNAs, circRNA, and lncRNA) transcripts involved in metabolism, immunity, and signal transduction in infertile liver were identified. In conclusion, this study shows that gonadal infertility is associated with not only changes in histological structure and hormone secretion but also changes in metabolism, immunity, and signal transduction networks in the liver. These results provide valuable information concerning the mechanism underlying infertility in fish.

Genome-Wide Analysis of the Expression of Circular RNA Full-Length Transcripts and Construction of the circRNA-miRNA-mRNA Network in Cervical Cancer.

Increasing evidence suggests that circular RNA (circRNA) plays an important role in tumorigenesis by regulating gene expression at the transcriptional and post-transcriptional levels. Alternative splicing events permit multiple transcript isoforms of circRNA to be produced; however, changes in the expression of circRNA full-length transcripts in cervical cancer remain unclear. Here, we systematically explored the dysregulation circRNA full-length transcripts and constructed an improved circRNA-miRNA-mRNA regulatory network to provide potential biomarkers and possible treatment targets in cervical cancer. We identified 9359 circular full-length transcripts from RNase R-treated RNA-seq data in cervical cancer, of which 353 circular full-length transcripts were significantly differentially expressed (DE) between the tumor and normal group. A total of 881 DE mRNA transcript isoforms were also identified from total RNA-seq data in cervical cancer, of which 421 (47.8%) transcript isoforms were up-regulated, and 460 (52.2%) transcript isoforms were down-regulated in tumor samples. Two circRNA-miRNA-mRNA competitively regulated networks, including 33 circRNA transcripts, 2 miRNAs, and 189 mRNA transcripts were constructed. Three genes (COPE, RAB3B, and TFPI) in the network were significantly associated with overall survival (P < 0.05), which indicated that these genes could act as prognostic biomarkers for patients with cervical cancer. Our study revealed genome-wide differential expression of full-length circRNA transcripts and constructed a more accurate circRNA-miRNA-mRNA network at the full-length transcript expression level in cervical cancer. CircRNA may thus be involved in the development of cervical cancer by regulating the expression of COPE, RAB3B, and TFPI. However, the specific regulatory mechanism in cervical cancer requires further study.

Revealing Epigenetic Factors of circRNA Expression by Machine Learning in Various Cellular Contexts.

SummaryCircular RNAs (circRNAs) have been identified as naturally occurring RNAs that are highly represented in the eukaryotic transcriptome. Although a large number of circRNAs have been reported, the underlying regulatory mechanism of circRNAs biogenesis remains largely unknown. Here, we integrated in-depth multi-omics data including epigenome, transcriptome, and non-coding RNA and identified candidate circRNAs in six cellular contexts. Next, circRNAs were divided into two classes (high versus low) with different expression levels. Machine learning models were constructed that predicted circRNA expression levels based on 11 different histone modifications and host gene expression. We found that the models achieve great accuracy in predicting high versus low expressed circRNAs. Furthermore, the expression levels of host genes of circRNAs, H3k36me3, H3k79me2, and H4k20me1 contributed greatly to the classification models in six cellular contexts. In summary, all these results suggest that epigenetic modifications, particularly histone modifications, can effectively predict expression levels of circRNAs.

Comprehensive analysis of circular RNA in oral leukoplakia: upregulated circHLA-C as a potential biomarker for diagnosis and prognosis.

BackgroundEmerging evidence indicates that circular RNAs (circRNAs) play an indispensable role in a variety of tumors, yet the function of circRNAs in premalignant lesions is still obscure. Oral leukoplakia (OLK) is one of the most common premalignant lesions of the oral mucosa. Our study aimed to comprehensively investigate whether circRNAs contribute to the occurrence and development of OLK.MethodsWe obtained six pairs of OLK and normal oral mucosal (NOM) tissue samples and subjected them to high-throughput sequencing to detect the expression of circRNA. In total, 26 pairs of NOM and OLK tissues were used for validation. Key circRNAs were selected and further validated by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), ribonuclease (RNase) R digestion, and Sanger sequencing. Visualization analysis of circular human leukocyte antigen-C (circHLA-C) was performed in the UCSC Genome Browser (genome.ucsc.edu). Functional analysis of differentially expressed (DE) circRNAs were processed by Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Furthermore, TargetScan (www.targetscan.org) was applied to predict targeted micro RNAs (miRNAs) and messenger RNAs (mRNAs) of circRNAs and a competing endogenous RNA (ceRNA) network related with identified circRNAs was constructed in Cytoscape (v2.8.0).ResultsProfile data showed that 366 circRNAs were significantly altered in OLK tissues, including 65 upregulated and 301 downregulated circRNA transcripts. Compared with sequencing results, seven selected circRNAs expressed the same changing tendency. The amplest upregulated circRNA in our sequencing data, circHLA-C, was confirmed through back-splice junction sequences by Sanger sequencing after RNase R digestion. Correlation analysis demonstrated that circHLA-C correlated positively with the degree of dysplasia. Furthermore, receiver operating characteristic (ROC) curve analysis indicated that circHLA-C had potential diagnostic value with excellent accuracy and specificity.ConclusionsAccording to the literature, we were the first to uncover the expression profiles of circRNAs in OLK. Our research performed a comprehensive bioinformatics analysis of DE circRNAs in OLK and identified circHLA-C as a promising diagnostic biomarker with potential as a therapeutic genetic target for OLK.

RNA-Seq Profiling of Serum Exosomal Circular RNAs Reveals Circ-PNN as a Potential Biomarker for Human Colorectal Cancer.

Circular RNAs (circRNAs) are emerging as cardinal areas of focus in the non-coding RNA field. Growing evidences have revealed exosomal circRNAs as potential biomarkers for detection of various cancers. However, the clinical importance of most serum exosomal circRNAs in colorectal cancer (CRC) have rarely been investigated. In this study, we examined the possible clinical application of serum exosomal circRNAs in the diagnosis of CRC. Firstly, we conducted RNA sequencing (RNA-seq) analysis using fifty CRC and fifty healthy control serum samples to identify CRC-related circRNAs. The sequencing data showed 122 differentially expressed circRNAs including 100 up-regulated and 22 down-regulated circRNA transcripts in CRC patients. Then, eight most dysregulated circRNAs were selected for validation by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay. Validation analysis revealed that the serum exosomal circ-PNN (hsa_circ_0101802) levels were significantly up-regulated in CRC cases compared with those in the healthy control groups. Receiver operating characteristic curve (ROC) analysis suggested that circ-PNN had significant value in CRC diagnosis with areas under the ROC curve (AUC) of 0.855 and 0.826 in the training and validation sets, respectively. We also found that the AUC of serum exosomal circ-PNN for early-stage CRC was 0.854. Finally, a network map based on circ-PNN was constructed to determine its potential miRNA-mRNAs binding. We also demonstrated that the expression of hsa-miR-6833-3P, hsa-let-7i-3p and hsa-miR-1301-3P were negatively correlated with circ-PNN in CRC patients. Collectively, our findings indicated that serum exosomal circ-PNN might be a potential non-invasive biomarker for the detection of CRC and may play a crucial role in the pathogenesis of CRC.