Circular RNA Expression Profiles Research Articles

Characterization of circRNA expression profiles associated with non-Mendelian inheritance in feather growth of chickens

ABSTRACT 1. Non-coding RNAs, such as miRNAs, play a crucial role in chicken feather growth rate. However, circular RNA (circRNA) expression profiles in fast- and slow-feathering chickens that follow and do not follow Mendelian inheritance are unclear. 2. The circRNA expression profiles was analysed by RNA sequencing of hair follicles of slow-feathering chickens that follow genetic rules and fast-feathering chickens that did not follow genetic rules. Differentially expressed circRNA-miRNA-mRNA competing endogenous RNA (ceRNA) network was then constructed and the key factors and regulation mechanisms controlling feather growth rate were identified. 3. The results revealed that 67 circRNAs were significantly differentially expressed in hens, including 22 up-regulated and 45 down-regulated circRNAs in non-Mendelian inheritance-mediated fast-feathering hens compared with Mendelian inheritance-mediated slow-feathering hens. In addition, 16 significantly differentially expressed circRNAs were identified in cockerels, including nine up-regulated and seven down-regulated circRNAs in non-Mendelian inheritance-mediated fast- compared with Mendelian inheritance-mediated slow-feathering cocks. Moreover, circRNA-mediated ceRNA regulation of hair follicle formation was particularly abundant in the Jak-STAT, Wnt and Toll-like receptor signalling pathways. Furthermore, circABI3BP was seen to be a crucial circRNA in regulating feather growth rate, by binding with gga-miR-1649-5p to regulate SSTR2 expression. 4. In conclusion, this study analysed circRNA expression profiles in fast- and slow-feathering chickens that follow and do not follow Mendelian inheritance, which laid the foundation for understanding the role of circRNA in chicken feather growth rate.

Circular RNA expression profiles and functional predication after restraint stress in the amygdala of rats.

Prolonged or repeated exposure to stress elevates the risk of various psychological diseases, many of which are characterized by central nervous system dysfunction. Recent studies have demonstrated that circular RNAs (circRNAs) are highly abundant in the mammalian brain. Although their precise expression and function remain unknown, they have been hypothesized to regulate transcriptional and post-transcriptional gene expression. In this investigation, we comprehensively analyzed whether restraint stress for 2 days altered the circRNA expression profile in the amygdala of male rats. The impact of restraint stress on behavior was evaluated using an elevated plus maze and open field test. Serum corticosterone levels were measured using an enzyme-linked immunosorbent assay. A total of 10,670 circRNAs were identified using RNA sequencing. Ten circRNAs were validated by reverse transcription and quantitative polymerase chain reaction analysis. Gene ontology and Kyoto encyclopedia of genes and genomes pathway analyzes supported the notion that genes associated with differentially expressed circRNAs are primarily implicated in neuronal activity and neurotransmitter transport. Moreover, the three differentially expressed circRNAs showed high specificity in the amygdala. Overall, these findings indicate that differentially expressed circRNAs are highly enriched in the amygdala and offer a potential direction for further research on restraint stress.

Alteration in the Expression of Circular Rnas and its association with the Development and Progression of Osteosarcoma, an Integrative Review with High Sensitivity Research.

Osteosarcoma is the most common primary malignant bone tumor, mainly affecting children, young adults, and the elderly. It is an aggressive cancer with a poor prognosis, exhibiting low survival rates even with standard treatment. Recently, circular RNA molecules capable of influencing gene expression through various functions, with their main role being acting as microRNA sponges and reducing their intracellular expression, have been identified. Recent studies have linked circular RNAs to osteosarcoma development and progression. Therefore, the present study aimed to investigate the alteration in circular RNA expression during osteosarcoma development and progression. An integrative literature review was conducted from September 10th to November 12th, 2021, using the following databases: PubMed/MEDLINE, SCOPUS, Web of Science, OVID, and EMBASE. 129 full articles were included in the review. The obtained data were organized using a standardized data collection instrument, which included the following information: altered expression profile of circular RNAs, associated cancer hallmarks, clinical-pathological relationships of circular RNAs, and perspectives on the studied circular RNAs. A total of 94 distinct circular RNAs were identified, predominantly showing an increased expression pattern. Approximately 91% of the studies that aimed to identify the mechanisms of action of circular RNAs highlighted the function of circular RNAs as microRNA sponges. The most associated cancer hallmarks with the identified circular RNAs were proliferative signaling induction, invasion and metastasis, and resistance to cell death. The altered expression of these circular RNAs generally correlated with a worse prognosis for patients, as evidenced by clinical features such as shorter survival, advanced Enneking and/or TNM stage, higher incidence of metastasis, larger tumor size, and increased chemoresistance. These findings indicate the significance of circular RNA molecules in osteosarcoma carcinogenesis, suggesting their potential as new prognostic and/or diagnostic biomarkers, as well as alternative therapeutic targets in the fight against osteosarcoma.

CircRNA circRREB1 promotes tumorigenesis and progression of breast cancer by activating Erk1/2 signaling through interacting with GNB4

Current investigations have illuminated the essential roles played by circular RNAs (circRNAs) in driving breast cancer (BC) tumorigenesis. However, the functional implications and molecular underpinnings of most circRNAs in BC are not well characterized. Here, Circular RNA (circRNA) expression profiles were analyzed in four surgically resected BC cases along with adjacent non-cancerous tissues applying RNA microarray analysis. The levels and prognostic implications of circRREB1 in BC were subjected to quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH). Experimental manipulation of circRREB1 levels in both in vivo and in vitro settings further delineated its role in BC cell growth, invasion, and metastasis. The mechanical verification of circRREB1's interaction with GNB4 was established through RNA pull-down, mass spectrometry, Western blot analysis, RNA immunoprecipitation assays (RIP), fluorescence ISH (FISH), and rescue experiments. We found that circRREB1 exhibited significant upregulation in BC tissues and cells, implicating its association with an unfavorable prognosis in BC patients. CircRREB1 knockdown elicited anti-proliferative, anti-migratory, anti-invasive, and pro-apoptotic effects in BC cells, whereas its upregulation exerted opposing influences. Follow-up mechanistic examinations suggested that circRREB1 might interact with GNB4 directly, inducing the activation of Erk1/2 signaling and driving BC progression. Our findings collectively indicate that the interplay of circRREB1 with GNB4 promotes Erk1/2 signaling, thereby fostering BC progression, and positioning circRREB1 as a candidate therapeutic target for intervention in BC.

Abstract 5680: Circular MALAT1 - the new kid on the block in breast-to-brain metastatic cancer

Abstract Introduction: Breast-to-brain metastatic (BTB met) cancer constitutes a great challenge for modern medicine and science. As the research on protein-coding genes brought only incremental progress in developing anticancer therapy, much attention is devoted to understanding the role of non-coding RNAs, including a relatively recently discovered group - of circular RNAs. This project aimed to determine the relationship between BTB met cancer cells and cells derived from a deadly brain tumor - glioblastoma stem-like cells (GSCs), considering the circular RNA signatures and selecting a reliable candidate for mechanistic study - a circular form of non-coding oncogene MALAT1 (circMALAT1). Methods: The circular RNA expression profile in patient-derived BTB met cancer cells (n=4), GSCs (n=8), and neural progenitor cells (NPC) as a non-malignant control (n=3) was evaluated by the microarray approach. Expression of circMALAT1 was also validated in non-malignant breast epithelial cells by digital PCR and data mining in the GEO database. CircMALAT1 knockdown by antisense oligonucleotides was followed by functional analysis. Results: 12,660 circular RNAs were detected using the Arraystar platform, and upon applying the cutoff of FC ≥2 and p-value <0.05, 547 circular RNAs were selected as significantly deregulated in all cancer groups compared to NPC. CircMALAT1 was elevated in BTB met compared to NPC and normal breast epithelial cells. Knockdown of circMALAT1 affected cancer cell viability and proliferation while promoting p21 expression, thus decelerating the cell cycle manifested by a reduction in 3D spheroids volume. Conclusions: The analysis of the circular RNA landscape showed for the first time some striking similarities between BTB mets and GSCs despite different tissue-of-origin. Of note, the circRNAome of BTB mets clustered especially closely with mesenchymal GSC - the most aggressive and therapy-resistant subtype of GSCs. These results also demonstrated the role of circMALAT1 in shaping the oncogenic traits of these cells. Therefore, circular RNAs can serve as valuable biomarkers and promising novel mechanistic candidates whose modulation may become a paradigm-shifting therapeutic approach. Citation Format: Adrian Szczepaniak, Agnieszka Bronisz, Jakub Godlewski. Circular MALAT1 - the new kid on the block in breast-to-brain metastatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5680.

Identification and characterization of circular RNAs expression profiles in obstructive sleep apnea-induced liver injury.

Circular RNAs (circRNAs) have exhibited microRNA sponge activity, related to many important biological processes. Our study attempted to explore the comprehensive changes of circRNAs expression pattern in Obstructive sleep apnea (OSA)-induced liver injury and provide a global perspective of differentially expressed circRNAs (DECs). Then, RT-qPCR was used to confirm the microarray data. Further, gene ontology (GO) and KEGG pathway analysis were performed to annotate the DECs. Finally, the circRNA-miRNA-mRNA interaction network was established to predicted the target genes and target miRNAs of DECs for a stepwise bioinformatics analysis. We revealed a total of eighty DECs. In the meantime, six circRNAs were randomly validated by RT-qPCR. Among these circRNAs, mmu_circRNA_000469, 37851, 38959, 38983, 31665 were up-regulated in both microarray and qRT-PCR tissues, while mmu_circRNA_27565 was down-regulated. GO analysis revealed that circRNAs-target genes were largely related to liver function process such as carboxylic acid metabolic process and negative regulation of mitochondrial membrane potential. Meanwhile, KEGG analysis found that there were 13 pathways related to these circRNAs- target genes. And the most enriched pathway was Natural killer cell mediated cytotoxicity, which strongly suggests that immune responses may be important for the process of OSA-induced liver injury. In addition, four significant DECs (mmu_circRNA_000469, 38959, 38983, 27565) and their target mRNA and target miRNAs were further selected to establish the regulation network. Our study revealed that circRNAs may play a crucial role in OSA-induced liver injury and thus mmu_circRNA_000469, 38959, 38983, 27565 may serve as biomarkers of biological process of OSA-induced liver injury.

Comprehensive exosomal microRNA profile and construction of competing endogenous RNA network in autism spectrum disorder: A pilot study.

Exosomes have been demonstrated to exert momentous roles in autism spectrum disorder (ASD). However, few studies have reported a correlation between exosomal microRNAs (miRNAs) and ASD. To date, our understanding of crucial competing endogenous RNA (ceRNA) networks in ASD remains limited. Herein, the exosomal miRNA profile in the peripheral blood of children with ASD and healthy controls was investigated and the level of immune cell infiltration in ASD was evaluated to determine the distribution of immune cell subtypes. Exosomes were isolated from the peripheral blood of ten children with ASD and ten healthy controls, and further identified using transmission electron microscopy and western blot analysis. RNA sequencing was conducted to investigate exosomal miRNA profiles in patients with ASD. The mRNA and circular RNA (circRNA) expression profiles were acquired from the Gene Expression Omnibus (GEO) database. Differentially expressed mRNAs (DEmRNAs), miRNAs (DEmiRNAs), and circRNAs (DEcircRNAs) were identified and ceRNA regulatory networks were constructed. Furthermore, the immune cell infiltration levels in patients with ASD were evaluated. Exosomes were spherical, approximately 100 nm in size, and were confirmed via western blot analysis using exosome-associated markers CD9, CD63, and CD81. Thirty-five DEmRNAs, 63 DEmiRNAs, and 494 DEcircRNAs were identified in patients with ASD. CeRNA regulatory networks, including 6 DEmRNAs, 14 DEmiRNAs, and 86 DEcircRNAs, were established. Correlation analysis indicated that leucine-rich glioma inactivated protein 1 (LGI1) expression was significantly positively correlated with the content of CD8+ T cells. Our findings may be conducive to offering novel insights into this disease and providing further evidence of transcriptomic abnormalities in ASD.

Circular RNA expression profile and functional analysis of circUvrag in light-induced photoreceptor degeneration.

Circular RNAs (circRNAs) are implicated in retinal pathophysiology; however, their expression profiles and functions in photoreceptor apoptosis are largely unknown. We explored circRNA-expression profiles and circUvrag (host gene: Uvrag, ultraviolet radiation resistance associated gene) function in light-induced photoreceptor apoptosis. Sprague-Dawley rats and 661 W photoreceptor cells were exposed to blue light to establish light-induced photoreceptor degeneration. Differentially expressed circRNAs were identified using microarrays. Potential functions of dysregulated circRNAs were analysed using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. CircUvrag expression and localization were evaluated using quantitative RT-PCR and fluorescence in situ hybridization, respectively. CircUvrag overexpression and knockdown were induced using a plasmid and a small interfering RNA, respectively, and retinal function and structure were assessed using scotopic electroretinography, haematoxylin-eosin staining, and TUNEL staining. Microglial migration was assessed using IBA1 immunostaining. The apoptosis ratio of photoreceptor cells in vitro was detected using flow cytometry. We identified 764 differentially expressed circRNAs, which were potentially related with the development of retinal structures, including neurons, dendrites, and synapses, and might participate in nervous-system pathophysiology. Light exposure enriched circUvrag in the cytoplasm of photoreceptors in the outer nuclear layer (ONL). CircUvrag knockdown decreased photoreceptor apoptosis and microglial migration to the ONL after light exposure, preserving ONL thickness and a-wave amplitude. In vitro, circUvrag knockdown inhibited photoreceptor apoptosis, although circUvrag overexpression slightly promoted photoreceptor apoptosis. CircUvrag knockdown attenuated light-induced photoreceptor apoptosis, and might be a potential target in retinal degeneration.

Investigation of Circular RNA Expression Profiles in Ultrasound-guided Incomplete Radiofrequency Ablation Transplanted Tumor Models of Human Liver Cancer.

Abnormally expressed circular RNAs (circRNAs) are associated with many diseases and have important biological effects on the regulation of gene expression. However, the circRNA expression profile in incomplete radiofrequency ablation (RFA)-treated liver cancer (LC) patients has not been characterized. This study investigated the potential biological effects of differentially expressed (DE) circRNAs in an incomplete RFA-treated transplantation tumor model of human LC. A circRNA microarray was utilized to analyze changes in the circRNA expression profiles. CircRNA host gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were also conducted using computational biology. Quantitative real-time PCR (qPCR) was also performed on the selected DE-circRNAs to verify the reliability of the microarray. The circRNA/miRNA interactions were predicted by Arraystar software and confirmed by a dual-luciferase assay. Following RFA incomplete ablation, 76 DE-circRNAs were detected (|fold change |>1.5, P-value < 0.05), 21 of which were upregulated and 55 of which were downregulated. Computational biological analysis revealed that the T-cell receptor signaling pathway was the most significantly enriched pathway of the genes related to altered expression, as indicated by enrichment of LCK, AKT3 and DLG1. PCR results for the upregulated hsa_circRNA_103595 and downregulated hsa_circRNA_001264 indicated that the circRNA microarray sequencing results were reliable. Double luciferase reporter assays confirmed that hsa-miR-185-3p was the target miRNA of hsa_circRNA_103595. The current study confirmed the changes in the expression profiles of circRNAs in tumor transplantation models after incomplete ablation, these changes may play a crucial role in the pathophysiological process of residual cancer transplantation tumors. These findings could lead to new directions for investigating the molecular biological mechanisms underlying RFA-treated LC as well as new ideas for treating LC by regulating circRNAs.

Specific Circular RNA Signature of Endothelial Cells: Potential Implications in Vascular Pathophysiology.

Circular RNAs (circRNAs) are a recently characterized family of gene transcripts forming a covalently closed loop of single-stranded RNA. The extent of their potential for fine-tuning gene expression is still being discovered. Several studies have implicated certain circular RNAs in pathophysiological processes within vascular endothelial cells and cancer cells independently. However, to date, no comparative study of circular RNA expression in different types of endothelial cells has been performed and analysed through the lens of their central role in vascular physiology and pathology. In this work, we analysed publicly available and original RNA sequencing datasets from arterial, veinous, and lymphatic endothelial cells to identify common and distinct circRNA expression profiles. We identified 4713 distinct circRNAs in the compared endothelial cell types, 95% of which originated from exons. Interestingly, the results show that the expression profile of circular RNAs is much more specific to each cell type than linear RNAs, and therefore appears to be more suitable for distinguishing between them. As a result, we have discovered a specific circRNA signature for each given endothelial cell type. Furthermore, we identified a specific endothelial cell circRNA signature that is composed four circRNAs: circCARD6, circPLXNA2, circCASC15 and circEPHB4. These circular RNAs are produced by genes that are related to endothelial cell migration pathways and cancer progression. More detailed studies of their functions could lead to a better understanding of the mechanisms involved in physiological and pathological (lymph)angiogenesis and might open new ways to tackle tumour spread through the vascular system.

Differences of circular RNA expression profiles between monozygotic twins' blood, with the forensic application in bloodstain and saliva

Monozygotic twins (MZTs) possess identical genomic DNA sequences and are usually indistinguishable through routine forensic DNA typing methods, which can be relevant in criminal and paternity cases. Recently, novel epigenetic methods involving DNA methylation and microRNA analysis have been introduced to differentiate MZTs. In this study, we explore the potential of using epigenetic markers, specifically circular RNAs (circRNAs), a type of non-coding RNA (ncRNA), to identify MZTs, and investigate the unique expression patterns of circRNAs within pairs of MZTs, enabling effective differentiation. Epigenetics regulates gene expression at the post-transcriptional level and plays a crucial role in cell growth and aging. CircRNAs, a recently characterized subclass of ncRNA, have a distinct covalent loop structure without the typical 5′ cap or 3′ tail. They have been reported to modulate various cellular processes and play roles in embryogenesis and eukaryotic development. To achieve this, we conducted a comprehensive circRNA sequencing analysis (circRNA-seq) using total RNA extracted from the blood samples of five pairs of MZTs. We identified a total of 15,257 circRNAs in all MZTs using circRNA-seq. Among them, 3, 21, 338, and 2967 differentially expressed circRNAs (DEcircRNAs) were shared among five, four, three, and two pairs of MZTs, respectively. Subsequently, we validated twelve selected DEcircRNAs using real-time quantitative polymerase chain reaction (RT-qPCR) assays, which included hsa_circ_0004724, hsa_circ_0054196, hsa_circ_004964, hsa_circ_0000591, hsa_circ_0005077, hsa_circ_0054853, hsa_circ_0054716, hsa_circ_0002302, hsa_circ_0004482, hsa_circ_0001103, novel_circ_0030288 and novel_circ_0056831. Among them, hsa_circ_0005077 and hsa_circ_0004482 exhibited the best performance, showing differences in 7 out of 10 pairs of MZTs. These twelve differentially expressed circRNAs also demonstrated strong discriminative power when tested on saliva samples from 10 pairs of MZTs. Notably, hsa_circ_0004724 displayed differential expression in 8 out of 10 pairs of MZTs in their saliva. Additionally, we evaluated the detection sensitivity, longitudinal temporal stability, and suitability for aged bloodstains of these twelve DEcircRNAs in forensic scenarios. Our findings highlight the potential of circRNAs as molecular markers for distinguishing MZTs, emphasizing their suitability for forensic application.

The circular RNA expression profile of human auricle cartilage and the role of circCOL1A2 in isolated microtia

Microtia is one of the most common craniofacial birth defects worldwide, and its primary clinical manifestation is auricle deformity. Epigenetic factors are known to contribute to the etiology of microtia, yet the involvement of circular RNAs (circRNAs) in human auricle development and their association with microtia remains poorly understood. In this study, we aimed to analyze differentially expressed circRNAs and explore their functional implications in isolated microtia. By employing circRNA microarray analysis and bioinformatics approaches, we identified 340 differentially expressed circRNAs in auricle cartilage of patients with isolated microtia, comprising 152 upregulated and 188 downregulated circRNAs. A circRNA-mRNA co-expression network was constructed, followed by gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Subsequently, we selected four significantly upregulated circRNAs from the co-expression network based on their association with cartilage development and validated their expressions in 30 isolated microtia and 30 control clinical auricle cartilage samples. Among these circRNAs, circCOL1A2, the most significantly upregulated circRNA, was selected as a representative circRNA for investigating its role in isolated microtia. Overexpression of circCOL1A2 significantly inhibited chondrocyte proliferation and chondrogenic differentiation of human mesenchymal stem cells. Additionally, circCOL1A2 upregulated Dermatan Sulfate Epimerase Like (DSEL) expression by sponging miR-637 through the competing endogenous RNA (ceRNA) mechanism. Notably, the downregulation of DSEL attenuated the inhibitory effect of circCOL1A2 overexpression on cell proliferation and chondrogenic differentiation. Collectively, these findings highlight the involvement of circCOL1A2 in the pathogenesis of isolated microtia and emphasize the potential significance of dysregulated circRNAs in disease development.

Identification of Differential Circular RNA Expression Profiles and Functional Networks in Human Macrophages Induced by Virulent and Avirulent Mycobacterium tuberculosis Strains.

Circular RNAs (circRNAs) are noncoding RNAs with diverse functions. However, most Mycobacterium tuberculosis (M.tb)-related circRNAs remain undiscovered. In this study, we infected THP-1 cells with virulent and avirulent M.tb strains and then sequenced the cellular circRNAs. Bioinformatic analysis predicted 58,009 circRNAs in all the cells. In total, 2035 differentially expressed circRNAs were identified between the M.tb-infected and uninfected THP-1 cells and 1258 circRNAs were identified in the virulent and avirulent M.tb strains. Further, the top 10 circRNAs were confirmed by Sanger sequencing, among which four circRNAs, namely circSOD2, circCHSY1, circTNFRSF21, and circDHTKD1, which were highly differentially expressed in infected cells compared with those in uninfected cells, were further confirmed by ring formation, specific primers, and RNase R digestion. Next, circRNA-miRNA-mRNA subnetworks were constructed, such as circDHTKD1/miR-660-3p/IL-12B axis. Some of the individual downstream genes, such as miR-660-3p and IL-12B, were previously reported to be associated with cellular defense against pathological processes induced by M.tb infection. Because macrophages are important immune cells and the major host cells of M.tb, these findings provide novel ideas for exploring the M.tb pathogenesis and host defense by focusing on the regulation of circRNAs during M.tb infection.

Comprehensive analysis of the circular RNA expression profile and circRNA–miRNA–mRNA network in the goat skin with divergent wool curvature

AbstractWool curvature is one of the most valuable characteristics of Zhongwei goat fur. As the goats grow, the curvature progressively diminishes, which has a substantial impact on the quality and market value of wool. Circular RNAs (circRNAs) are a class of noncoding RNA and play vital roles in animal growth and development. However, the expression and function of circRNAs in the wool curvature of Zhongwei goats are unclear. In our study, we conducted circRNA expression profiling of Zhongwei goat skin exhibiting divergent curvature wool phenotypes at two developmental stages using the RNA‐seq. In total, 12,682 circRNAs and 158 differentially expressed circRNAs (DE circRNAs) were identified. KEGG analysis illustrated that host genes of DE circRNAs were significantly enriched in the signaling pathways of Ras, JAK/STAT5, and cAMP, which might affect wool curvature. We further validated five circRNAs using qRT‐PCR, which were consistent with the sequencing results. Functional verification assay demonstrated that circRNA8782 regulated fibroblast proliferation. In addition, we constructed a regulatory competing endogenous RNA (ceRNA) network and predicted circRNA3173‐miR‐16b‐5p‐IGF1 axes involved in the regulation of wool curvature. Our result will provide the foundation for uncovering the regulatory mechanisms of underlying wool curvature patterns in goats.

The identification and functional analysis of CircRNAs in endometrial receptivity of mice with polycystic ovary.

The disorders of endometrial receptivity and ovulatory dysfunction are both significant causes of infertility in patients with polycystic ovary syndrome (PCOS). In this study, we investigated the expression profile and functional implications of circular RNAs (circRNAs) in the endometrial receptivity of PCOS-affected mice. Twenty-four female C57BL/6 mice were divided into PCOS and normal control groups. The PCOS group received subcutaneous DHEA treatment, while the control group remained untreated. Gene chip technology was utilized to analyze circRNA expression in endometrial tissues on the fourth day of gestation with subsequent bioinformatics analyses into circRNA functions. Furthermore, endometrial epithelial cells were used to determine represented circRNA functions. Results showed that the PCOS group exhibited 205 differentially expressed circRNAs, with 147 upregulated and 58 downregulated ones. qRT-PCR confirmed differential expression of circRNAs, including circRNA_38548, circRNA_001686, circRNA_38550, and circRNA_27938. Predicted target genes and a circRNA-miRNA-mRNA regulatory network were constructed. Additionally, four circRNAs (circRNA_38548, circRNA_38550, and circRNA_001686) were identified to contribute to abnormal endometrial receptivity by regulating genes such as Lifr, FOXK1, FOXO1, HOXA10, through interactions with miRNAs. Further research is warranted to elucidate the underlying mechanisms involving these circRNAs.

Exosome‑delivered miR‑486‑3p inhibits the progression of osteosarcoma via sponging CircKEAP1/MARCH1 axis components.

Accumulating evidence shows that the disruption of competing endogenous RNA (ceRNA) networks plays a significant role in osteosarcoma (OS) initiation and progression. However, the specific roles and functions of the ceRNAs in OS remain unclear. First, differentially expressed microRNAs (DEMs) were identified by mining the E-MTAB-1136 and GSE28423 datasets. MiRWalk website was used to predict the target gene of miRNA. OS-associated circular RNA (circRNA) expression profiles were downloaded from the published microarray databases. Gene expression levels were assessed through reverse transcription-quantitative PCR and western blotting. The biological effects of circKEAP1, microRNA (miR)-486-3p and membrane-associated RINGCH finger protein 1 (MARCH1) in OS cells were investigated using Cell Counting Kit-8, Transwell, colony formation and wound healing assays. miR-486-3p was aberrantly downregulated in OS tissues and cell lines and was packed with exosomes. miR-486-3p overexpression was shown to inhibit OS cell progression and promoted cell cycle arrest in vitro. In addition, MARCH1 was identified as a direct downstream molecule of miR-486-3p in OS cells. circKEAP1 was found to be upregulated in OS tissues and cells. circKEAP1 was found to have binding sites with miR-486-3p. Mechanistically, circKEAP1 positively regulated MARCH1 expression by sponging miR-486-3p. Exosomal miR-486-3p inhibited the progression of OS by sponging the circKEAP1/MARCH1 axis. These findings may provide a promising treatment approach for OS.

Regulation of cashmere fineness traits by noncoding RNA in Jiangnan cashmere goats

BackgroundCashmere has long been used as the raw material for wool textiles. The diameter of the cashmere fibre determines its quality and economic value. However, the regulatory role of noncoding RNAs (ncRNAs) in cashmere fineness remains unclear, especially regarding the interaction between ncRNAs and coding RNAs.ResultsTranscriptome sequencing was used to identify the expression profiles of long noncoding RNAs (lncRNAs), circular RNAs (circRNAs) and microRNAs (miRNAs) in the skin tissues of Jiangnan cashmere goats with different cashmere fineness levels. Integration analysis of ncRNA and coding RNA was performed in combination with previous research results. The results showed that 16,437 lncRNAs, 2234 circRNAs, and 1322 miRNAs were identified in 8 skin samples of cashmere goats. A total of 403 differentially expressed (DE) lncRNAs, 62 DE circRNAs and 30 DE miRNAs were identified in the skin tissues of the fine groups (Fe) and coarse groups (Ce). We predicted the target gene of DE lncRNA, the target gene of DE miRNA and the host gene of DE circRNA. Based on functional annotation and enrichment analysis of target genes, we found that DE lncRNAs could be involved in regulating the fineness traits of cashmere. The most potential lncRNAs were MSTRG.42054.1, MSTRG.18602.3, and MSTRG.2199.13.ConclusionsThe data from this study enriched the cashmere goat noncoding RNA database and helped to supplement the annotation of the goat genome. The results provided a new direction for the breeding of cashmere characters.

Research on Circular RNA Expression Profiles in the Photoaging Mouse Model.

Nude mouse has been widely used to study photoaging induced by long-term chronic UV exposure. Circular RNAs (circRNAs) have been previously identified in several diseases. However, the roles of circRNAs in photoaging and potential regulatory mechanisms remain unclear. To identify specific circRNAs differentially expressed in photoaged skin and investigate their potential role in aging. In this study, we screened out the microarray data to profile the expression of circRNAs. The circRNAs were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway. 36 circRNAs were identified to be differentially expressed between the UV group and control group (fold change > 1.5; P < 0.05), including 6 upregulated and 30 downregulated circRNAs. GO and KEGG biological pathway analyses indicated that the changes in circRNAs were associated with cancer, inflammation, oxidative stress, and metabolism. This present study revealed a circRNAs expression profiling in vivo. These findings not only provide a new possibility to prevent the occurrence of photoaging but also have therapeutic values for photoaging and associated skin diseases.

Characterization of circRNAs in established osimertinib‑resistant non‑small cell lung cancer cell lines.

Drug resistance is an urgent problem to be solved in the treatment of non‑small‑cell lung cancer (NSCLC). Osimertinib is a third‑generation EGFR‑tyrosine kinase inhibitor, which can improve the efficacy and quality of life of patients; however, the inevitable resistance after long‑term use of osimertinib often leads to treatment failure. Cell lines are key tools for basic and preclinical studies. At present, few osimertinib‑resistant cell lines (HCC827‑OR and H1975‑OR) have been established. In the present study, osimertinib‑resistant cell lines were established by gradually increasing the drug concentration. Half‑maximal inhibitory concentration (IC50), cell morphology, whole exon sequencing, Cell Counting Kit‑8 assay, EdU staining and flow cytometry were used to evaluate the osimertinib‑resistant cell lines. Western blot analysis was used to detect the expression levels of key proteins involved in osimertinib resistance. The circular RNA (circRNA) expression profile was identified by RNA sequencing (RNA‑seq) analysis of HCC827, HCC827‑OR, H1975 and H1975‑OR cells. Subsequently, the biological roles of differentially expressed circRNAs were explored in invitro studies. Osimertinib‑resistant cell lines were successfully established via treatment with an increasing concentration of osimertinib. Osimertinib IC50 and proliferation of resistant cells were much higher than those of sensitive cells. Notably, phosphorylated (p)‑AKT and p‑ERK were markedly activated in resistant cells, and the inhibitory effect of osimertinib on p‑AKT and p‑ERK was weaker in resistant cells than that in parental cells. RNA‑seq analysis identified differentially expressed circRNAs in HCC827, HCC827‑OR, H1975 and H1975‑OR cells. The most dysregulated circRNAs (circPDLIM5 and circPPP4R1) were selected for further functional study. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the host genes of differentially expressed circRNAs were associated with 'endocytosis' and 'regulation of autophagy'. In conclusion, the present study established osimertinib‑resistant cell lines and revealed that circRNAs may serve as a promising biomarker in NSCLC osimertinib resistance.

Expression profile of circular RNA in inflammatory response in human bronchial epithelial cells induced by carbon black nanoparticles

Objective: To explore the toxic effect of carbon black nanoparticles on human bronchial epithelial cells, and identify the differentially expressed circular RNA based on the full transcriptome high-throughput sequencing, so as to provide evidence for the development of biomarkers exposed to carbon black nanoparticles and their application on epigenetic toxicology. Methods: In June 2020, 16 HBE cells were treated with carbon black nanoparticles at concentrations of 20, 40 and 80 μg/ml, and 16 HBE cells without any intervention were used as the control group. The cytotoxicity of carbon black nanoparticles was detected by CCK8 and LDH experiments. Real-time quantitative fluorescent PCR (qRT-PCR) and ELISA were used to detect the changes of interleukin-6 (IL-6) and interleukin-8 (IL-6, IL-8) mRNA and protein levels of carbon black nanoparticles with concentration gradient after 72 h exposure. Western blot analysis was conducted to detect the expression levels of toll-like receptor 4 (TLR4), phosphorylated nuclear factor-κB (P-NF-κB), apoptosis-related speckled protein (ASC) and Caspase-1 associated with nuclear factor-κB. According to high-throughput sequencing results, differentially expressed Circrnas were screened and identified by qRT-PCR, and those with stable differentially expressed circrnas and the strongest association with the NF-κB pathway were selected for ring performance identification. Results: After being exposed to carbon black nanoparticles for 72 h, the activity of 16HBE cells decreased significantly (P<0.05), and the release of lactate dehydrogenase increased significantly (P<0.05). Compared with control group, mRNA expression levels of IL-6 and IL-8, protein levels of IL-6 and IL-8 were increased, and protein levels of TLR4, p-NF-κB, ASC and Caspase-1 were significantly up-regulated in 16 HBE cells of different concentrations, with statistical significance (P<0.05). Compared with the control group, a total of 492 differentially expressed circular Rnas (|log2 FC|>1) were detected. Among the 5 differentially expressed (P<0.05) circular Rnas, circ_002642 was selected as the object of subsequent research on circular Rnas, affter 72 hours of exposure to 80 μg/ml CBNPs, 16HBE cells showed signlficantly higher expression of circ_002642 (P<0.05) . Conclusion: Carbon black nanoparticles can induce differentially expressed circular RNAs associated with inflammatory response in human bronchial epithelial cells.