Differentially Expressed circRNAs Research Articles

Comprehensive evaluation of circRNAs in cirrhotic cardiomyopathy before and after liver transplantation.

Cirrhotic cardiomyopathy (CCM) is a common complication of liver cirrhosis. Many patients with cirrhotic livers do not die from liver failure but from abnormal hemodynamics secondary to liver cirrhosis. Liver transplantation is one of the most effective treatments for liver diseases. Recent studies have found that liver transplantation can reverse CCM and improve cardiac function; however, its role and remedial mechanism remain unclear. Circular RNAs (circRNAs) have become an important marker for diagnosing diseases. The differential expression of circRNAs is associated with heart diseases. In this study, we used gene sequencing to detect the circRNA expression profile of patients with CCM before and after liver transplantation and predicted the differential circRNA target genes. The results showed that a total of 1495 circRNAs were dysregulated after liver transplantation, 1319 genes were downregulated, and 176 were upregulated (P<0.05, log2 (fold change)>2.0). The qRT-PCR results showed that circ-ASAP1, circ-N4BP2L2, circ-EXOC6B were significantly downregulated (P<0.05), which were consistent with the RNA sequencing data, and circ-ASAP1 had the most significant difference. Bioinformatics analysis suggested that mTOR and MAPK signaling pathways might be involved in the pathogenesis of CCM. By constructing a circRNA-miRNA-mRNA interaction network, hsa-miR-197-3p, hsa-miR-483-3p, and hsa-miR-885-3p, particularly key miRNA (hsa-miR-483-3p), were found to be the major potential genes involved in CCM regulation. In summary, this study suggested that circRNAs play a crucial regulatory role in the occurrence of CCM before and after liver transplantation, and their potential biological function might be the key to diagnosis and treatment.

The circular RNA landscape in multiple sclerosis: Disease-specific associated variants and exon methylation shape circular RNA expression profile.

Circular RNAs (circRNAs) are a class of non-coding RNAs increasingly emerging as crucial actors in the pathogenesis of human diseases, including autoimmune and neurological disorders as multiple sclerosis (MS). Despite several efforts, the mechanisms regulating circRNAs expression are still largely unknown and the circRNA profile and regulation in MS-relevant cell models has not been completely investigated. In this work, we aimed at exploring the global landscape of circRNA expression in MS patients, also evaluating a possible correlation with their genetic and epigenetic background. We performed RNA-seq experiments on circRNA-enriched samples, derived from peripheral blood mononuclear cells (PBMCs) of 10 MS patients and 10 matched controls and performed differential circRNA expression. The genetic background was evaluated using array genotyping, and an expression quantitative trait loci (eQTL) analysis was carried out. Expression analysis revealed 166 differentially expressed circRNAs in MS patients, 125 of which are downregulated. One of the top dysregulated circRNAs, hsa_circ_0007990, derives from the PGAP3 gene, encoding a protein relevant for the control of autoimmune responses. The downregulation of this circRNA was confirmed in two independent replication cohorts, suggesting its implementation as a possible RNA-based biomarker. The eQTL analysis evidenced a significant association between 89 MS-associated loci and the expression of at least one circRNA, suggesting that MS-associated variants could impact on disease pathogenesis by altering circRNA profiles. Finally, we found a significant correlation between exon methylation and circRNA expression levels, supporting the hypothesis that epigenetic features may play an important role in the definition of the cell circRNA pool. We described the circRNA expression profile of PBMCs in MS patients, suggesting that MS-associated variants may tune the expression levels of circRNAs acting as "circ-QTLs", and proposing a role for exon-based DNA methylation in regulating circRNA expression.

Analyses of circRNAs profiles of the lactating and nonlactating crops in pigeon (Columba livia)

Pigeon has the specific biological ability to produce pigeon milk (also known as crop milk) by its crop. Circular RNAs (circRNAs) are important noncoding RNAs acting as the sponges of miRNAs, but the molecular mechanism of circRNAs regulating crop milk production has not been reported in pigeon. We compared expression profiles of crops during lactating and nonlactating crops, and networks of competing endogenous RNAs (ceRNAs) were constructed. The results showed a total of 8,723 circRNAs were identified, and there were 770 differentially expressed circRNAs (DECs) between these two different periods of crops. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the host genes of DECs were enriched in GnRH, MAPK, Insulin, Wnt, and AMPK signaling pathways. Furthermore, gga_circ_0000300 interacted with miR-92-2-5p, which targeted genes participating in lactation and milk composition synthesis. Gga_circ_0003018, gga_circ_0003019 and gga_circ_0003020 could bind with let-7c-5p regulating SOCS3 in crop milk production. These findings provide the circRNAs expression profiles and facilitate the analysis of molecular mechanism of crop milk production in pigeon.

Circular RNA CircSLC8A1 contributes to osteogenic differentiation in hBMSCs via CircSLC8A1/miR-144-3p/RUNX1 in periprosthetic osteolysis.

Circular RNAs (circRNAs) are often found in eukaryocyte and have a role in the pathogenesis of a variety of human disorders. Our related research has shown the differential expression of circRNAs in periprosthetic osteolysis (PPOL). However, the involvement of circRNAs in the exact process is yet unknown. CircSLC8A1 expression was evaluated in clinical samples and human bone marrow mesenchymal stem cells (hBMSCs) in this investigation using quantitative real-time PCR. In vitro and in vivo studies were conducted to explicate its functional role and pathway. We demonstrated CircSLC8A1 is involved in PPOL using gain- and loss-of-function methods. The association of CircSLC8A1 and miR-144-3p, along with miR-144-3p and RUNX1, was predicted using bioinformatics. RNA pull-down and luciferase assays confirmed it. The impact of CircSLC8A1 in the PPOL-mouse model was also investigated using adeno-associated virus. CircSLC8A1 was found to be downregulated in PPOL patients' periprosthetic tissues. Overexpression of CircSLC8A1 promoted osteogenic differentiation (OD) and inhibited apoptosis of hBMSCs in vitro. The osteogenic markers of RUNX1, osteopontin (OPN) and osteocalcin (OCN) were significantly upregulated in hBMSCs after miR-144-3p inhibitor was transferred. Mechanistic analysis demonstrated that CircSLC8A1 directly bound to miR-144-3p and participated in PPOL through the miR-144-3p/RUNX1 pathway in hBMSCs. Micro-CT and quantitative analysis showed that CircSLC8A1 markedly inhibited PPOL, and osteogenic markers (RUNX1, OPN and OCN) were significantly increased (P<0.05) in the mice model. Our findings prove that CircSLC8A1 exerted a regulatory role in promoting osteogenic differentiation in hBMSCs, and CircSLC8A1/miR-144-3p/RUNX1 pathway may provide a potential target for prevention of PPOL.

Transcriptome-based insights into the regulatory role of immune-responsive circular RNAs in Litopanaeus vannamei upon WSSV infection

Circular RNAs (circRNAs) are non-coding RNAs (ncRNAs) originating from a post-transcriptional modification process called back-splicing. Despite circRNAs being traditionally considered by-products rather than independently functional, circRNAs play many vital roles, such as in host immunity during viral infection. However, in shrimp, these remain largely unexplored. Therefore, this study aims to identify circRNAs in Litopenaeus vannamei in the context of WSSV infection, one of the most eradicative pathogens threatening shrimp populations worldwide. We identified 290 differentially expressed circRNAs (DECs) in L. vannamei upon WSSV infection. Eight DECs were expressed from their parental genes, including alpha-1-inhibitor-3, calpain-B, integrin-V, hemicentin-2, hemocytin, mucin-17, proPO2, and rab11-FIP4. These were examined quantitatively by qRT-PCR, which revealed the relevant expression profiles to those obtained from circRNA-Seq. Furthermore, the structural and chemical validation of the DECs conformed to the characteristics of circRNAs. One of the functional properties of circRNAs as a miRNA sponge was examined via the interaction network between DECs and WSSV-responsive miRNAs, which highlighted the targets of miRNA sponges. Our discovery could provide insight into the participation of these ncRNAs in shrimp antiviral responses.

Transcriptome analysis of differentially expressed circular RNAs in the testis and ovary of golden pompano (Trachinotus blochii)

The artificial breeding of golden pompano (Trachinotus blochii) has expanded greatly in recent years, and after long-term breeding efforts, clear sexual dimorphisms have been observed in T. blochii growth traits, with females growing faster. As sponges of microRNA (miRNAs), circular RNAs (CircRNAs) can alleviate miRNA inhibition of target mRNA. However, few studies have examined sex-related CircRNAs and none of those have looked at T. blochii. To further understand the role of CircRNAs in sex differentiation and sexual size dimorphism in T. blochii, six CircRNA libraries were constructed from the testes and ovaries of T. blochii. A total of 1522 CircRNAs were found distributed over all 24 chromosomes of T. blochii. 135 differentially expressed CircRNAs (DECs) were identified by screening, These DECs were then subjected to GO enrichment, which found 47 enriched pathways. A number of CircRNAs were enriched in cellular processes and metabolic processes. According to the KEGG pathway analysis, a series of sex differentiation pathways were enriched, including the GnRH, calcium, and MAPK signaling pathways. Furthermore, we selected two CircRNAs from the DECs named circ-cacna1b and circ-octc. We found that the cacna1b gene is regulated by 7 miRNAs, 3 of which were regulated by circ-cacna1b, i.e., mmu-miR-138-5p, fru-miR-138, and pma-miR-138b. In addition, the miRNA named pma-miR-138b can regulate sex-related genes, such as sox9 and dmrt1, among others. The co-expression network of CircRNA-miRNA-mRNA showed circ-cacna1b may play a crucial role in T. blochii sex differentiation by regulating pma-miR-138b to affect the expression of sex differentiation genes. The circ-octc may be one of the largest contributors to sexual size dimorphism during growth through its effect on lipid metabolism. These findings could broaden our understanding of CircRNAs and provide new insight into their function in sex differentiation and growth.

Whole transcriptome sequencing and competitive endogenous RNA regulation network construction analysis in benzo[a]pyrene-treated breast cancer cells

Breast cancer is the most common malignant tumor in women worldwide, and environmental pollutants are considered to be risk factors. Currently, most studies into benzo[a]pyrene (B[a]P)-induced breast cancer focus on biological effects such as proliferation, invasion, and metastasis, DNA damage, estrogen receptor (ER)-related molecular mechanisms, oxidative damage, and other metabolic pathways. This study aims to provide insights into the role of B[a]P in breast cancer development through RNA-seq and bioinformatics analysis and construction of a competing endogenous RNA (ceRNA) regulatory network. By analyzing RNA-seq results, we identified 144 differentially-expressed circRNAs, 69 differentially-expressed lncRNAs, 20 differentially-expressed miRNAs, and 212 differentially-expressed mRNAs. Following on, we analyzed the gene ontology (GO) and KEGG enrichment functions of the differentially-expressed RNAs. In addition, the protein-protein interaction (PPI) network was mapped for differentially-expressed mRNAs. Subsequently, we constructed ceRNA networks, one of which consisted of 45 dysregulated circRNAs, 11 miRNAs, and 9 mRNAs, and a second consisted of 40 lncRNAs, 11 miRNAs, and 9 mRNAs. Finally, 6 circRNAs, 4 lncRNAs, 1 miRNA, and 4 mRNAs were randomly selected for quantitative real-time PCR verification. PCR results were further verified by Western blotting assays. These results show that the expression level of differentially-expressed RNA was consistent with the sequencing data, and the Western blotting results were highly consistent with the PCR results, confirming that the sequencing result was very reliable. This study systematically explores the ceRNA atlas of differentially-expressed genes related to B[a]P exposure in breast cancer cells, providing new insights into mechanisms of environmental pollutants in breast cancer.

Analysis of circRNA and miRNA expression profiles in IGF3-induced ovarian maturation in spotted scat (Scatophagus argus).

Insulin-like growth factor 3 (IGF3) induces ovarian maturation in teleosts; however, research on its molecular regulatory mechanism remains deficient. Circular RNAs (circRNAs) and microRNAs (miRNAs) are involved in various biological processes, including reproduction. In this study, circRNAs and miRNAs involved in IGF3-induced ovarian maturation were evaluated in spotted scat (Scatophagus argus). In ovarian tissues, we identified 176 differentially expressed (DE) circRNAs and 52 DE miRNAs between IGF3 treatment and control groups. Gene Ontology (GO) enrichment analyses showed that host genes of DE circRNAs and target genes of DE miRNAs were enriched for various processes with a high degree of overlap, including cellular process, reproduction, reproductive process, biological adhesion, growth, extracellular region, cell junction, catalytic activity, and transcription factor activity. Enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways included cell adhesion molecules, ECM-receptor interaction, regulation of actin cytoskeleton, focal adhesion, cell cycle, Hedgehog signaling pathway, phosphatidylinositol signaling system, PI3K-Akt signaling pathway, Apelin signaling pathway, Notch signaling pathway, insulin signaling pathway, and Rap1 signaling pathway. A circRNA-miRNA-mRNA regulatory network was constructed, including DE genes involved in reproduction (e.g., oocyte maturation, oocyte meiosis, and ECM remodeling), such as ccnd2, hecw2, dnm2, irs1, adam12, and cdh13. According to the regulatory network and tissue distribution, we identified one circRNA (Lachesis_group5:6245955|6270787) and three miRNAs (novel_miR_622, novel_miR_980, and novel_miR_64) that may exert regulatory effects in IGF3-induced ovarian maturation in S. argus. Taken together, this study provides a novel insight into the molecular mechanisms by which IGF3 functions in ovaries and highlights the effects of circRNAs and miRNAs in reproduction in S. argus.

Whole-transcriptome sequencing revealed differentially expressed mRNAs and non-coding RNAs played crucial roles in NiONPs-induced liver fibrosis

Nickel oxide nanoparticles (NiONPs) induced liver fibrosis, while its mechanisms associated with transcriptome remained unclear. This study aimed to investigate the roles of differentially expressed (DE) messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs) in NiONPs-induced liver fibrosis, and further confirm whether JNK/c-Jun pathway enriched by the DE RNAs was involved in the regulation of the disease. A liver fibrosis rat model was established by intratracheal perfusion of NiONPs twice a week for 9 weeks. Whole-transcriptome sequencing was applied to obtain expression profiles of mRNAs, long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs) in the model rat and control liver tissues. Comparing the RNA expression profiles of the model and control liver tissues, we identified 324 DE mRNAs, 129 DE lncRNAs, 24 DE miRNAs and 33 DE circRNAs, and the potential interactions among them were revealed by constructing two co-expression networks, including lncRNA–miRNA–mRNA and circRNA–miRNA–mRNA networks. Using RT-qPCR, we verified the sequencing results of some RNAs in the networks and obtained similar expression profiles, indicating our sequencing results were reliable and referable. Through Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, we predicted the biological functions and signaling pathways potentially related to NiONPs-induced liver fibrosis, such as “positive regulation of JNK cascade”, “inflammatory response”, “transcription factor binding”, and MAPK, Wnt, PI3K-Akt signaling pathways. JNK/c-Jun pathway, a subclass of MAPK signal, was selected for further investigation because it was significantly enriched by fibrosis-related DE genes and activated in animal models. In vitro, we detected the cytotoxicity of NiONPs on LX-2 cells and treated the cells with 5 μg/ml NiONPs for 12 h. The results showed NiONPs induced the up-regulated protein expression of fibrotic factors collagen-1a1 (Col-1a1) and matrix metalloproteinas2 (MMP2) and JNK/c-Jun pathway activation. While these effects were reversed after JNK/c-Jun pathway was blocked by SP600125 (JNK pathway inhibitor), indicating the pathway was involved in NiONPs-induced excessive collagen formation. In conclusion, our results revealed the DE mRNAs and ncRNAs played crucial roles in NiONPs-induced liver fibrosis, and JNK/c-Jun pathway mediated the development of the disease.

Transcriptome Sequencing Analysis of circRNA in Skeletal Muscle between Fast- and Slow-Growing Chickens at Embryonic Stages.

Skeletal muscle growth has always been the focus of the broiler industry, and circRNAs play a significant role in this process. We collected leg muscles of slow- and fast-growing Bian chicken embryos in the study at 14 (S14 and F14) and 20 (S20 and F20) days for RNA-seq. Finally, 123 and 121 differentially expressed circRNAs (DECs) were identified in S14 vs. F14 and S20 vs. F20, respectively. GO enrichment analysis for DECs obtained important biological process (BP) terms including nicotinate nucleotide biosynthetic process, nicotinate nucleotide salvage, and NAD salvage in S20 vs. F20 and protein mannosylation in S14 vs. F14. KEGG pathway analysis showed Wnt signaling pathway, Tight junction, Ubiquitin mediated proteolysis, and Notch signaling pathway were enriched in the top 20. Based on the GO and KEGG analysis results, we found some significant host genes and circRNAs such as NAPRT and novel_circ_0004547, DVL1 and novel_circ_0003578, JAK2 and novel_circ_0010289, DERA and novel_circ_0003082, etc. Further analysis found 19 co-differentially expressed circRNAs between the two comparison groups. We next constructed a circRNA-miRNA network for them, and some candidate circRNA-miRNA pairs related to skeletal muscle were obtained, such as novel_circ_0002153-miR-12219-5p, novel_circ_0003578-miR-3064-3p, and novel_circ_0010661-miR-12260-3p. These results would help to reveal the mechanism for circRNAs in skeletal muscle and also provide some guidance for the breeding of broilers.

Identification and characterization of circRNAs related to meat quality during embryonic development of the longissimus dorsi muscle in two pig breeds.

Meat quality, an important economic trait, is regulated by many factors, especially by genetic factors, including coding genes, miRNAs, and lncRNAs. Recent studies have elucidated that circRNAs also play a key role in muscle development and lipid deposition. However, the functions and regulatory mechanisms of circRNAs in meat quality remain mostly unknown. The circRNA expression profiles between Huainan pigs (Chinese indigenous pigs, fat-type, Huainan HN) and Large White pigs (Western commercial pigs, lean-type, LW) in the longissimus dorsi (LD) muscle at 38, 58, and 78days post conception (dpc) were compared by sequencing. In total, 39,887 circRNAs were identified in 18 samples, and 60, 78, and 86 differentially expressed circRNAs (DECs) were found at the three stages mentioned above between these two breeds. The parent genes of DECs were enriched in myogenesis, proliferation, adipogenesis and muscle fiber-type transition. The circRNA-miRNA interaction networks included 38 DECs and 47 miRNAs, and these miRNAs were involved in muscle development and lipid metabolism. Two shared DECs (circ_0030593 and circ_0032760) of these three stages were selected, their head-to-tail junction sites were validated by Sanger sequencing, and RT‒qPCR results suggested that these two DECs might be involved in intramuscular fat deposition. These findings provide a basis for understanding the role of circRNAs in meat quality.

Screening and analysis of differentially expressed circRNAs and miRNAs in chronic diabetic extremity wounds.

Increasing studies have shown that circular RNAs (circRNAs) and microRNAs (miRNAs) are related to the development of endocrine and metabolic diseases. However, there are few reports on the expression of circRNAs and miRNAs and their related co-expression and the expression of competitive endogenous RNA (ceRNA) in diabetic chronic refractory wounds. In this study, we compared the differential expression of circRNAs and miRNAs in diabetes chronic refractory wounds and normal skin tissues by high-throughput gene sequencing, and screened the differentially expressed circRNAs and miRNAs. Five abnormally expressed circRNAs and seven abnormally expressed miRNAs were detected by reverse transcription quantitative polymerase chain reaction PCR (RT-qPCR)to verify the results of RNA sequencing. We applied gene ontology (GO) to enrich and analyze dysregulated genes and elucidated their main functions via the Kyoto encyclopedia of genes and genomes analysis (KEGG). We constructed coding noncoding gene co-expression networks and ceRNA networks based on significantly abnormally expressed genes. According to the results of coding noncoding gene co-expression network analysis, hsa_circRNA_104175, hsa_circRNA_ 001588, hsa_circRNA_104330, hsa_circRNA_ 100141, hsa_circRNA_103107, and hsa_ circRNA_102044 may be involved in the regulation of the chronic intractable wound healing process in diabetes mellitus. This is particularly true in the regulation of vascular smooth muscle contraction-related pathways and the actin cytoskeleton, which affect the healing of chronic intractable wounds in diabetes. MiR-223-5p, miR-514a-3p, miR-205-5p, and miR-203-3p, which each have a targeting relationship with the above circRNAs, regulate the metabolism of nitrogen compounds in wound tissue by regulating NOD-like receptor signaling pathways, signaling pathways regulating the pluripotency of stem cells, microRNAs in cancer, and ECM-receptor interaction. This study showed circRNAs, miRNAs, and their network are associated with the development of chronic intractable wounds in diabetes, and our research identified the goals for new molecular biomarkers and gene therapy.

Identification and differential analysis of noncoding RNAs in response to drought in Phyllostachys aureosulcata f. spectabilis.

The role of noncoding RNAs (ncRNAs) in plant resistance to abiotic stresses is increasingly being discovered. Drought stress is one of the most common stresses that affecting plant growth, and high intensity drought has a significant impact on the normal growth of plants. In this study, a high-throughput sequencing was performed on plant tissue samples of Phyllostachys aureosulcata f. spectabilis C. D. Chu et C. S. Chao by drought treatment for 0, 2, 4 and 6 days. The sequencing results were analysed bioinformatically. We detected 336,946 RNAs among all 12 samples, including 192,098 message RNAs (mRNAs), 142,761 long noncoding RNAs (lncRNAs), 1,670 circular RNAs (circRNAs), and 417 microRNAs (miRNAs). We detected 2,419 differentially expressed (DE) ncRNAs, including 213 DE circRNAs, 2,088 DE lncRNAs and 118 DE miRNAs. Then, we used Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) to functionally predict DE ncRNAs. The results showed that most DE ncRNAs are involved in the response to drought stress, mainly in biochemical reactions involved in some metabolites, as well as in organelle activities. In addition, we validated two random circRNAs and demonstrated their circularity. We also found a stable internal reference gene available for Phyllostachys aureosulcata f. spectabilis and validated the accuracy of this experiment by quantitative real-time polymerase chain reaction (qRT-PCR).

Hsa_circ_0074158 regulates the endothelial barrier function in sepsis and its potential value as a biomarker.

Background: Sepsis is one of the main causes of death in critically ill patients with high morbidity and mortality. Circular RNAs (CircRNAs) are aberrantly expressed, and play significant regulatory roles in many diseases. However, the expression profiles and functions of circRNAs in sepsis have not yet been fully clarified. Methods: Our present study performed an RNA sequencing (RNA-seq) analysis to assess the expression profiles of circRNAs in vitro. We applied the quantitative real-time polymerase chain reaction (RT-qPCR) to verify the RNA-seq results. The analyses of Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, the competitive endogenous RNA (ceRNA) regulatory networks, were performed to explore the potential mechanism in sepsis. And then, significantly up-regulated differentially expressed (DE) circRNA, hsa_circ_0074158, was selected for further study. Hsa_circ_0074158 was silenced to investigate its regulatory function in sepsis, and the barrier function was also examined in vitro. Endothelial cell junctions were valued using Vascular endothelial cadherin (VE-cadherin), which was detected by immunofluorescence staining. We measured endothelial permeability by transendothelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran extravasation. Results: In total, 203 significantly DE circRNAs, including 77 up-regulated and 126 down-regulated, were identified. In vitro, the RT-qPCR assay showed that the expression pattern of hsa_circ_0074158, hsa_circ_RSBN1L_11059, hsa_circ_0004188, and hsa_circ_0005564 were consistent with the results from RNA-seq analysis. The expression of hsa_circ_0074158 detected by RT-qPCR in vivo was also consistent with the RNA-seq results. The ceRNA networks, GO enrichment, and the KEGG pathway analyses revealed that circRNAs may be related to the barrier function in sepsis. The immunofluorescence assay showed that the suppression of hsa_circ_0074158 expression significantly enhanced the expression of VE-cadherin, which was suppressed in lipopolysaccharide (LPS)-induced sepsis. Additionally, hsa_circ_0074158 knockdown could partially reverse the LPS-induced TEER reduction and FITC-dextran extravasation elevation in sepsis. Conclusion: In conclusion, we have found DE circRNAs could serve as potential biomarkers and therapeutic targets for sepsis. Hsa_circ_0074158 plays a vital role in sepsis and is related to the disruption of the endothelial barrier.

MicroRNA and circular RNA profiling in the deposited fat tissue of Sunite sheep.

As the most typical deposited fat, tail fat is an important energy reservoir for sheep adapted to harsh environments and plays an important role as a raw material in daily life. However, the regulatory mechanisms of microRNA (miRNA) and circular RNA (circRNA) in tail fat development remain unclear. In this study, we characterized the miRNA and circRNA expression profiles in the tail fat of sheep at the ages of 6, 18, and 30 months. We identified 219 differentially expressed (DE) miRNAs (including 12 novel miRNAs), which exhibited a major tendency to be downregulated, and 198 DE circRNAs, which exhibited a tendency to be upregulated. Target gene prediction analysis was performed for the DE miRNAs. Functional analysis revealed that their target genes were mainly involved in cellular interactions, while the host genes of DE circRNAs were implicated in lipid and fatty acid metabolism. Subsequently, we established a competing endogenous RNA (ceRNA) network based on the negative regulatory relationship between miRNAs and target genes. The network revealed that upregulated miRNAs play a leading role in the development of tail fat. Finally, the ceRNA relationship network with oar-miR-27a_R-1 and oar-miR-29a as the core was validated, suggesting possible involvement of these interactions in tail fat development. In summary, DE miRNAs were negatively correlated with DE circRNAs during sheep tail fat development. The multiple ceRNA regulatory network dominated by upregulated DE miRNAs may play a key role in this developmental process.

Differential Expression and Bioinformatics Analysis of Plasma-Derived Exosomal circRNA in Type 1 Diabetes Mellitus.

Both exosome and circular RNA (circRNA) have been reported to participate in the pathogenesis of type 1 diabetes mellitus (T1DM). However, the exact role of exosomal circRNA in T1DM is largely unknown. Here, we identified the exosomal circRNA expression profiles in the plasma of T1DM patients and explored their potential function using bioinformatics analysis. Material and Methods. Exosomes were extracted by the size exclusion chromatography method from plasma of 10 T1DM patients and 10 age- and sex- matched control subjects. Illumina Novaseq6000 platform was used to detect the exosomal circRNA expression profiles. Multiple bioinformatics analysis was applied to investigate the potential biological functions of exosomal circRNAs. A total of 784 differentially expressed exosomal circRNAs have been identified in T1DM patients, of which 528 were upregulated and 256 were downregulated. Gene Ontology analysis enriched terms such as protein ubiquitination involved in ubiquitin-dependent protein catabolic protein (GO:0042787), membrane (GO:0016020), and GTPase activator activity (GO:0005096). The most enriched pathway in Kyoto Encyclopedia of Genes and Genomes was ubiquitin-mediated proteolysis (ko04120). The miRNA-targeting prediction method was used to identify the miRNAs that bind to circRNAs, and circRNA-miRNA-mRNA pathways were constructed, indicating that interactions between circRNA, miRNA, and gene might be involved in the disease progression. The present study identified the exosomal circRNA expression profiles in T1DM for the first time. Our results threw novel insights into the molecular mechanisms of T1DM.

In Silico Identification and Characterization of circRNAs During Host-Pathogen Interactions.

Circular RNAs (circRNAs) are a class of non-coding RNAs that are formed via back-splicing. These circRNAs are predominantly studied for their roles as regulators of various biological processes. Notably, emerging evidence demonstrates that host circRNAs can be differentially expressed (DE) upon infection withpathogens (e.g., influenza and coronaviruses), suggesting a role for circRNAs in regulating host innate immune responses. However, investigations on the role of circRNAs during pathogenic infections are limited by the knowledge and skills required to carry out the necessary bioinformatic analysis to identify DE circRNAs from RNA sequencing (RNA-seq) data. Bioinformatics prediction and identification of circRNAs is crucial before any verification, and functional studies using costly and time-consuming wet-lab techniques. To solve this issue, a step-by-step protocol of in silico prediction and characterization of circRNAs using RNA-seq data is provided in this manuscript. The protocol can be divided into four steps: 1) Prediction and quantification of DE circRNAs via the CIRIquant pipeline; 2) Annotation via circBase and characterization of DE circRNAs; 3) CircRNA-miRNA interaction prediction through Circr pipeline; 4) functional enrichment analysis of circRNA parental genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). This pipeline will be useful in driving future in vitro and in vivo research to further unravel the role of circRNAs in host-pathogen interactions.

High-throughput data on circular RNA reveal novel insights into chronic glomerulonephritis.

Circular RNAs (circRNAs), a unique novel type of RNA, have been widely reported to be involved in physiologic and pathologic processes in humans. However, the exact molecular pathogenesis of circRNAs in chronic glomerulonephritis (CGN) is far from clear. This paper aims to evaluate the specific expression profile of circRNAs in renal cortex tissues from Adriamycin-induced CGN rats. CircRNAs were screened in renal cortex tissues from 3 CGN rats and 3 control rats by using high-throughput sequencing (HTS). Then, 4 circRNAs were selected randomly for verification by quantitative real-time polymerase chain reaction (qRT-PCR). In addition, the differentially expressed (DE) circRNAs were analyzed by bioinformatics methods. In total, 31 significantly DE circRNAs were identified, which revealed their potential roles in CGN; in particular, we found that 4 confirmed altered circRNAs (rno-circ-RNAs 689, 3217, 1327, and 5001) might play important roles in the development of CGN. This study reveals a cluster of circRNAs that are DE in Adriamycin-induced CGN rats, which brings us closer to understanding the pathogenic mechanisms and may provide new potential targets for clinical treatment.

Pro-Inflammatory Cytokines Promote the Transcription of Circular RNAs in Human Pancreatic β Cells.

Circular RNAs (circRNAs) have recently been implicated in impaired β-cell function in diabetes. Using microarray-based profiling of circRNAs in human EndoC-βH1 cells treated with pro-inflammatory cytokines, this study aimed to investigate the expression and possible regulatory roles of circRNAs in human β cells. We identified ~5000 β-cell-expressed circRNAs, of which 84 were differentially expressed (DE) after cytokine exposure. Pathway analysis of the host genes of the DE circRNAs revealed the enrichment of cytokine signaling pathways, indicative of circRNA transcription from inflammatory genes in response to cytokines. Multiple binding sites for β-cell-enriched microRNAs and RNA-binding proteins were observed for the highly upregulated circRNAs, supporting their function as ‘sponges’ or ‘decoys’. We also present evidence for circRNA sequence conservation in multiple species, the presence of cytokine-induced regulatory elements, and putative protein-coding potential for the DE circRNAs. This study highlights the complex regulatory potential of circRNAs, which may play a crucial role during immune-mediated β-cell destruction in type 1 diabetes.

CircNf1-mediated CXCL12 expression in the spinal cord contributes to morphine analgesic tolerance

BackgroundSevere pain in patients can be alleviated by morphine treatment. However, long-term morphine treatment induces analgesic tolerance and the molecular mechanism of morphine analgesic intolerance is still not fully elucidated. Therefore, a novel target for improving morphine analgesic tolerance is required. Whole-genome sequencing showed that circNf1 is highly expressed in the dorsal horns of morphine-treated rats. Circular RNAs (circRNAs) are known to be unique and conserved cellular molecules that are mostly present in cytoplasm and participate in various biochemical processes with different functions. Therefore, we focused on exploring the molecular mechanism by which circNf1 contributes to morphine analgesic tolerance. MethodsCircRNA sequencing revealed differential expression of circRNAs after morphine treatment, and bioinformatics software programs (miRNAda, PicTar, and RNAhybrid) were used to predict possible mRNAs and binding sites. RNA binding protein immunoprecipitation (RIP), chromatin isolation by RNA purification (ChIRP), fluorescence in situ hybridization (FISH), western blotting, biotin-coupled probe pull-down assay, luciferase assay, and quantitative real-time polymerase chain reaction (qRT-PCR) were conducted to detect and measure the expression levels of circRNAs, mRNAs, and proteins. Intrathecal injections of small interfering RNAs (siRNAs), microRNA (miRNA) agomirs, and functional virus microinjections were administered to artificially mediate the expression of molecules. Tail immersion and hotplate tests were performed to evaluate morphine analgesic tolerance. ResultsMorphine-induced circNf1 expression was high in the spinal cord. RIP-PCR and luciferase assay data showed that circNf1 could combine with both miR-330-3p and miR-665, and FISH showed that circNf1 co-localized with miR-330-3p and miR-665. qRT-PCR assay showed downregulation of miR-330-3p and miR-665 in morphine-treated rats; western blotting results showed that CXCL12 increased after morphine treatment, however, the upregulation of CXCL12 could be alleviated after the intrathecal injection of miR-330-3p as well as miR-665 agomir. qRT-PCR indicated that circNf1 can bind to CXCL12 promoter, the increased circNf1 can enhance CXCL12 mRNA in naïve rats, and inhibition of circNf1 can alleviate the upregulation of CXCL12 mRNA in morphine-treated rats. Behavioral tests revealed that inhibition of circNf1 and CXCL12 and the enhancement of miR-330-3p and miR-665 can alleviate morphine analgesic tolerance. ConclusionsOur study indicates a novel pathway that can contribute to morphine analgesic tolerance, the circRNA to cytokine pathway, in which circNf1 functions as a sponge for miR-330-3p and miR-665 and induces the upregulation of CXCL12 at both transcriptional and translational levels in morphine-treated rats.