Differential expression of circRNAs during osteogenic/odontogenic differentiation of stem cells from apical papilla promoted by blue light-emitting diode.

BACKGROUND: Circular RNA (circRNA) is a key player in regulating the multidirectional differentiation of stem cells. Previous research by our group found that the blue light-emitting diode (LED) had a promoting effect on the osteogenic/odontogenic differentiation of human stem cells from apical papilla (SCAPs). This research aimed to investigate the differential expression of circRNAs during the osteogenic/odontogenic differentiation of SCAPs regulated by blue LED. MATERIALS AND METHODS: SCAPs were divided into the irradiation group (4 J/cm2) and the control group (0 J/cm2), and cultivated in an osteogenic/odontogenic environment. The differentially expressed circRNAs during osteogenic/odontogenic differentiation of SCAPs promoted by blue LED were detected by high-throughput sequencing, and preliminarily verified by qRT-PCR. Functional prediction of these circRNAs was performed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the circRNA-miRNA-mRNA networks were also constructed. RESULTS: It showed 301 circRNAs were differentially expressed. GO and KEGG analyses suggested that these circRNAs were associated with some signaling pathways related to osteogenic/odontogenic differentiation. And the circRNA-miRNA-mRNA networks were also successfully constructed. CONCLUSION: CircRNAs were involved in the osteogenic/odontogenic differentiation of SCAPs promoted by blue LED. In this biological process, circRNA-miRNA-mRNA networks served an important purpose, and circRNAs regulated this process through certain signaling pathways.

Current advances in circular RNA detection and investigation methods: Are we running in circles?

Circular RNAs (circRNAs), characterized by their closed-loop structure, have emerged as significant transcriptomic regulators, with roles spanning from microRNA sponging to modulation of gene expression and potential peptide coding. The discovery and functional analysis of circRNAs have been propelled by advancements in both experimental and bioinformatics tools, yet the field grapples with challenges related to their detection, isoform diversity, and accurate quantification. This review navigates through the evolution of circRNA research methodologies, from early detection techniques to current state-of-the-art approaches that offer comprehensive insights into circRNA biology. We examine the limitations of existing methods, particularly the difficulty in differentiating circRNA isoforms and distinguishing circRNAs from their linear counterparts. A critical evaluation of various bioinformatics tools and novel experimental strategies is presented, emphasizing the need for integrated approaches to enhance our understanding and interpretation of circRNA functions. Our insights underscore the dynamic and rapidly advancing nature of circRNA research, highlighting the ongoing development of analytical frameworks designed to address the complexity of circRNAs and facilitate the assessment of their clinical utility. As such, this comprehensive overview aims to catalyze further advancements in circRNA study, fostering a deeper understanding of their roles in cellular processes and potential implications in disease. This article is categorized under: RNA Methods > RNA Nanotechnology RNA Methods > RNA Analyses in Cells RNA Methods > RNA Analyses In Vitro and In Silico.

The role of circular RNAs in regulating resistance to cancer immunotherapy: mechanisms and implications.

Cancer immunotherapy has rapidly transformed cancer treatment, yet resistance remains a significant hurdle, limiting its efficacy in many patients. Circular RNAs (circRNAs), a novel class of non-coding RNAs, have emerged as pivotal regulators of gene expression and cellular processes. Increasing evidence indicates their involvement in modulating resistance to cancer immunotherapy. Notably, certain circRNAs function as miRNA sponges or interact with proteins, influencing the expression of immune-related genes, including crucial immune checkpoint molecules. This, in turn, shapes the tumor microenvironment and significantly impacts the response to immunotherapy. In this comprehensive review, we explore the evolving role of circRNAs in orchestrating resistance to cancer immunotherapy, with a specific focus on their mechanisms in influencing immune checkpoint gene expression. Additionally, we underscore the potential of circRNAs as promising therapeutic targets to augment the effectiveness of cancer immunotherapy. Understanding the role of circRNAs in cancer immunotherapy resistance could contribute to the development of new therapeutic strategies to overcome resistance and improve patient outcomes.

Exosome-transported circ_0061407 and circ_0008103 play a tumour-repressive role and show diagnostic value in non-small-cell lung cancer.

BACKGROUND: Circular RNAs (circRNAs), one of the major contents of exosomes, have been shown to participate in the occurrence and progression of cancers. The role and the diagnostic potential of exosome-transported circRNAs in non-small-cell lung cancer (NSCLC) remain largely unknown. METHODS: The NSCLC-associated exosomal circ_0061407 and circ_0008103 were screened by circRNA microarray. The role of circ_0061407 and circ_0008103 in NSCLC was examined in vitro and in vivo. The encapsulation of the two circRNAs into exosomes and the transport to recipient cells were observed by confocal microscopy. The effects of exosome-transported circ_0061407 and circ_0008103 on recipient cells were investigated using a co-culture device. Bioinformatics analyses were performed to predict the mechanisms by which circ_0061407 and circ_0008103 affected NSCLC. The quantitative polymerase chain reaction was used to quantify the exosome-containing circ_0061407 and circ_0008103 in the serum samples of healthy, pneumonia, benign lung tumours, and NSCLC. The diagnostic efficacy was evaluated using receiver operating characteristic curves. RESULTS: The levels of circ_0061407 and circ_0008103 within exosomes were down-regulated in the serum of patients with NSCLC. The up-regulation of circ_0061407 and circ_0008103 inhibited the proliferation, migration/invasion, cloning formation of NSCLC cells in vitro and inhibited lung tumour growth in vivo. Circ_0061407 and circ_0008103 were observed to be packaged in exosomes and transported to recipient cells, where they inhibited the proliferation, migration/invasion, and cloning formation abilities of the recipient cells. Moreover, circ_0061407 and circ_0008103 might be involved in the progression of NSCLC by interacting with microRNAs and proteins. Additionally, lower serum exosomal circ_0061407 and circ_0008103 levels were associated with advanced pathological staging and distant metastasis. CONCLUSIONS: This study identified two novel exosome-transported circRNAs (circ_0061407 and circ_0008103) associated with NSCLC. These findings may provide additional insights into the development of NSCLC and potential diagnostic biomarkers for NSCLC.

Non-Coding RNA as Biomarkers and Their Role in the Pathogenesis of Gastric Cancer-A Narrative Review.

Non-coding RNAs (ncRNAs) represent a broad family of molecules that regulate gene expression, including microRNAs, long non-coding RNAs and circular RNAs, amongst others. Dysregulated expression of ncRNAs alters gene expression, which is implicated in the pathogenesis of several malignancies and inflammatory diseases. Gastric cancer is the fifth most frequently diagnosed cancer and the fourth most common cause of cancer-related death. Studies have found that altered expression of ncRNAs may contribute to tumourigenesis through regulating proliferation, apoptosis, drug resistance and metastasis. This review describes the potential use of ncRNAs as diagnostic and prognostic biomarkers. Moreover, we discuss the involvement of ncRNAs in the pathogenesis of gastric cancer, including their interactions with the members of major signalling pathways.

Circular RNAs in EMT-driven metastasis regulation: modulation of cancer cell plasticity, tumorigenesis and therapy resistance.

The non-coding RNAs comprise a large part of human genome lack of capacity in encoding functional proteins. Among various members of non-coding RNAs, the circular RNAs (circRNAs) have been of importance in the pathogenesis of human diseases, especially cancer. The circRNAs have a unique closed loop structure and due to their stability, they are potential diagnostic and prognostic factors in cancer. The increasing evidences have highlighted the role of circRNAs in the modulation of proliferation and metastasis of cancer cells. On the other hand, metastasis has been responsible for up to 90% of cancer-related deaths in patients, requiring more investigation regarding the underlying mechanisms modulating this mechanism. EMT enhances metastasis and invasion of tumor cells, and can trigger resistance to therapy. The cells demonstrate dynamic changes during EMT including transformation from epithelial phenotype into mesenchymal phenotype and increase in N-cadherin and vimentin levels. The process of EMT is reversible and its reprogramming can disrupt the progression of tumor cells. The aim of current review is to understanding the interaction of circRNAs and EMT in human cancers and such interaction is beyond the regulation of cancer metastasis and can affect the response of tumor cells to chemotherapy and radiotherapy. The onco-suppressor circRNAs inhibit EMT, while the tumor-promoting circRNAs mediate EMT for acceleration of carcinogenesis. Moreover, the EMT-inducing transcription factors can be controlled by circRNAs in different human tumors.

Circular RNAs: a small piece in the heart failure puzzle.

Cardiovascular disease, specifically heart failure (HF), remains a significant concern in the realm of healthcare, necessitating the development of new treatments and biomarkers. The RNA family consists of various subgroups, including microRNAs, PIWI-interacting RNAs (piRAN) and long non-coding RNAs, which have shown potential in advancing personalized healthcare for HF patients. Recent research suggests that circular RNAs, a lesser-known subgroup of RNAs, may offer a novel set of targets and biomarkers for HF. This review will discuss the biogenesis of circular RNAs, their unique characteristics relevant to HF, their role in heart function, and their potential use as biomarkers in the bloodstream. Furthermore, future research directions in this field will be outlined. The stability of exosomal circRNAs makes them suitable as biomarkers, pathogenic regulators, and potential treatments for cardiovascular diseases such as atherosclerosis, acute coronary syndrome, ischemia/reperfusion injury, HF, and peripheral artery disease. Herein, we summarized the role of circular RNAs and their exosomal forms in HF diseases.

M2 macrophage-derived exosomal circTMCO3 acts through miR-515-5p and ITGA8 to enhance malignancy in ovarian cancer.

Tumor-associated macrophages of the M2 phenotype promote cancer initiation and progression. Importantly, M2 macrophage-derived exosomes play key roles in the malignancy of cancer cells. Here, we report that circTMCO3 is upregulated in ovarian cancer patients, and its high expression indicates poor survival. M2-derived exosomes promote proliferation, migration, and invasion in ovarian cancer, but these effects are abolished by knockdown of circTMCO3. Furthermore, circTMCO3 functions as a competing endogenous RNA for miR-515-5p to reduce its abundance, thus upregulating ITGA8 in ovarian cancer. miR-515-5p inhibits ovarian cancer malignancy via directly downregulating ITGA8. The decreased oncogenic activity of circTMCO3-silencing exosomes is reversed by miR-515-5p knockdown or ITGA8 overexpression. Exosomal circTMCO3 promotes ovarian cancer progression in nude mice. Thus, M2 macrophage-derived exosomes promote malignancy by delivering circTMCO3 and targeting the miR-515-5p/ITGA8 axis in ovarian cancer. Our findings not only provide mechanistic insights into ovarian cancer progression, but also suggest potential therapeutic targets.

ALKBH5-mediated m6A modification of circFOXP1 promotes gastric cancer progression by regulating SOX4 expression and sponging miR-338-3p.

Circular RNAs (circRNAs) have recently been suggested as potential functional modulators of cellular physiology processes in gastric cancer (GC). In this study, we demonstrated that circFOXP1 was more highly expressed in GC tissues. High circFOXP1 expression was positively associated with tumor size, lymph node metastasis, TNM stage, and poor prognosis in patients with GC. Cox multivariate analysis revealed that higher circFOXP1 expression was an independent risk factor for disease-free survival (DFS) and overall survival (OS) in GC patients. Functional studies showed that increased circFOXP1 expression promoted cell proliferation, cell invasion, and cell cycle progression in GC in vitro. In vivo, the knockdown of circFOXP1 inhibited tumor growth. Mechanistically, we observed ALKBH5-mediated m6A modification of circFOXP1 and circFOXP1 promoted GC progression by regulating SOX4 expression and sponging miR-338-3p in GC cells. Thus, our findings highlight that circFOXP1 could serve as a novel diagnostic and prognostic biomarker and potential therapeutic target for GC.

Moxibustion ameliorates chronic inflammatory visceral pain via spinal circRNA-miRNA-mRNA networks: a central mechanism study.

This study aimed to unveil the central mechanism of moxibustion treating chronic inflammatory visceral pain (CIVP) from the angle of circRNA-miRNA-mRNA networks in the spinal cord. The rat CIVP model was established using a mixture of 5% (w/v) 2,4,6-trinitrobenzene sulfonic acid and 50% ethanol at a volume ratio of 2:1 via enema. Rats in the moxibustion group received herb-partitioned moxibustion at Tianshu (ST25, bilateral) and Qihai (CV6) points. The abdominal withdrawal reflex (AWR), mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL) were adopted for pain behavior observation and pain sensitivity assessment. The circRNA, miRNA, and mRNA expression profiles were detected using the high-throughput sequencing technique. Relevant databases and bioinformatics analysis methods were used to screen for differentially expressed (DE) RNAs and build a circRNA-miRNA-mRNA (competing endogenous RNA) ceRNA regulatory network. The real-time quantitative PCR was employed to verify the sequencing result. CIVP rat models had a significantly higher AWR and lower TWL and MWT than normal rats. Between normal and model rats, there were 103 DE-circRNAs, 16 DE-miRNAs, and 397 DE-mRNAs in the spinal cord. Compared with the model group, the moxibustion group had a lower AWR and higher TWL and MWT; between these two groups, there were 118 DE-circRNAs, 15 DE-miRNAs, and 804 DE-mRNAs in the spinal cord. Two ceRNA networks were chosen to be verified. As a result, moxibustion's analgesic effect on visceral pain in CIVP rats may be associated with regulating the circRNA_02767/rno-miR-483-3p/Gfap network in the spinal cord and improving central sensitization.

circ_0000592 facilitates the progression of esophageal squamous cell carcinoma via miR-155-5p/FZD5 axis.

Esophageal squamous cell carcinoma (ESCC) is one of the gastrointestinal malignancies with high prevalence and poor prognosis. Previous reports suggested that circular ribose nucleic acids might exert regulatory functions in ESCC. This study aims to explore the role of circ_0000592 in ESCC progression, providing novel insights into the diagnosis and therapeutic avenues for ESCC. The GSE131969 data set was utilized to assess circ_0000592 expression in ESCC. The validation was performed in the tumorous tissues of ESCC patients (n = 80) and human-immortalized ESCC cell lines. The correlation between circ_0000592 expression and prognosis was analyzed. The impact of circ_0000592 on ESCC cell activity was evaluated through downregulating circ_0000592, as well as encompassing cell viability, migration, and invasion abilities. The downstream pathway of circ_0000592 was explored by binding site prediction from the TargetScan database, followed by in vitro and in vivo experiments. An in vivo xenograft tumor model was established to highlight the role of circ_0000592 in ESCC. Patients with ESCC exhibited higher circ_0000592 expression levels compared to noncancerous patients, which were associated with reduced survival time, higher TNM stage, and increased lymph node metastasis. The circ_0000592 downregulation suppressed cell viability, migration, and invasion abilities in vitro. Mechanistically, circ_0000592 countered the inhibitory effects on the target gene Frizzled 5 (FZD5) through interactions with miR-155-5p. The overexpression of miR-155-5p curtailed the luciferase activity of circ_0000592 in ESCC cells, inhibiting downstream molecule FZD5 protein expression and subsequently mitigating the detrimental consequences of escalated circ_0000592 expression in ESCC cells. Consistently, circ_0000592 downregulation curbed proliferation and metastasis of ESCC tumors in vivo. In summary, circ_0000592 promoted the progress of ESCC by counteracting the inhibitory impact on FZD5 through its interaction with miR-155-5p. Together, our findings highlighted circ_0000592 as a prospective therapeutic target for ESCC.

CircSCNN1A inhibits the proliferation, migration and invasion of renal cell carcinoma cells by decreasing CLDN8 expression through miR-590-5p.

BACKGROUND: Increasing evidence suggests that circular RNA (circRNA) plays a regulatory role in the progression of renal cell carcinoma (RCC). However, the precise function and underlying mechanism of circSCNN1A in RCC progression still remain unclear. METHODS: The expression levels of circSCNN1A, microRNA-590-5p (miR-590-5p), claudin 8 (CLDN8), cyclin D1, matrix metalloprotein 2 (MMP2), MMP9, E-cadherin, N-cadherin and vimentin were detected by a quantitative real-time polymerase chain reaction and Western blotting analysis. Immunohistochemistry assay was performed to analyze the positive expression rate of CLDN8. Cell proliferation was investigated by cell colony formation, 5-Ethynyl-2'-deoxyuridine and DNA content quantitation assays. Cell migration and invasion were assessed by wound-healing and transwell invasion assays. Interactions among circSCNN1A, miR-590-5p and CLDN8 were identified by dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay. Xenograft mouse model assay was conducted to verify the effect of circSCNN1A on tumor formation in vivo. RESULTS: CircSCNN1A and CLDN8 expression were significantly downregulated, while miR-590-5p was upregulated in both RCC tissues and cells. CircSCNN1A overexpression inhibited RCC cell proliferation, migration and invasion, accompanied by decreases of cyclin D1, MMP2, MMP9, N-cadherin and vimentin expression and an increase of E-cadherin expression. CircSCNN1A acted as a miR-590-5p sponge and regulated RCC cell processes by binding to miR-590-5p. CLDN8, a target gene of miR-590-5p, was involved in the regulation of the biological behaviors of RCC cells by miR-590-5p. In addition, circSCNN1A induced CLDN8 production by interacting with miR-590-5p. Further, circSCNN1A suppressed tumor formation in vivo. CONCLUSION: CircSCNN1A inhibited RCC cell proliferation, migration and invasion by regulating the miR-590-5p/CLDN8 pathway.

Elucidating the Role of circTIAM1 in Guangling Large-Tailed Sheep Adipocyte Proliferation and Differentiation via the miR-485-3p/PLCB1 Pathway.

Fat tissue-a vital energy storage organ-is intricately regulated by various factors, including circular RNA, which plays a significant role in modulating fat development and lipid metabolism. Therefore, this study aims to clarify the regulatory mechanism of sheep adipocyte proliferation and differentiation by investigating the involvement of circTIAM1, miR-485-3p, and its target gene PLCB1. Through previous sequencing data, circTIAM1 was identified in sheep adipocytes, with its circularization mechanism elucidated, confirming its cytoplasmic localization. Experimental evidence from RNase R treatment and transcription inhibitors highlighted that circTIAM1 is more stable than linear RNA. Additionally, circTIAM1 promoted sheep adipocyte proliferation and differentiation. Furthermore, bioinformatic analysis demonstrated a robust interaction between miR-485-3p and circTIAM1. Further experiments revealed that miR-485-3p inhibits fat cell proliferation and differentiation by inhibiting PLCB1, with circTIAM1 alleviating the inhibitory effect via competitive binding. In summary, our findings elucidate the mechanism through which circTIAM1 regulates Guangling Large-Tailed sheep adipocyte proliferation and differentiation via the miR-485-3p-PLCB1 pathway, offering a novel perspective for further exploring fat metabolism regulation.

Identification of circRNA Expression Profile and Potential Systemic Immune Imbalance Modulation in Premature Rupture of Membranes.

Premature rupture of membrane (PROM) refers to the rupture of membranes before the onset of labor which increases the risk of perinatal morbidity and mortality. Recently, circular RNAs (circRNAs) have emerged as promising regulators of diverse diseases. However, the circRNA expression profiles and potential circRNA-miRNA-mRNA regulatory mechanisms in PROM remain enigmatic. In this study, we displayed the expression profiles of circRNAs and mRNAs in plasma and fetal membranes of PROM and normal control (NC) groups based on circRNA microarray, the Gene Expression Omnibus database, and NCBI's Sequence Read Archive. A total of 1,459 differentially expressed circRNAs (DECs) in PROM were identified, with 406 upregulated and 1,053 downregulated. Then, we constructed the circRNA-miRNA-mRNA network in PROM, encompassing 22 circRNA-miRNA pairs and 128 miRNA-mRNA pairs. Based on the analysis of gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and gene set enrichment analysis (GSEA), DECs were implicated in immune-related pathways, with certain alterations persisting even postpartum. Notably, 11 host genes shared by DECs of fetal membrane tissue and prenatal plasma in PROM were significantly implicated in inflammatory processes and extracellular matrix regulation. Our results suggest that structurally stable circRNAs may predispose to PROM by mediating systemic immune imbalances, including peripheral leukocyte disorganization, local immune imbalance at the maternal-fetal interface, and local collagen disruption. This is the first time to decipher a landscape on circRNAs of PROM, reveals the pathogenic cause of PROM from the perspective of circRNA, and opens up a new direction for the diagnosis and treatment of PROM.

Exosome-derived circUPF2 enhances resistance to targeted therapy by redeploying ferroptosis sensitivity in hepatocellular carcinoma.

BACKGROUND: Advanced hepatocellular carcinoma (HCC) can be treated with sorafenib, which is the primary choice for targeted therapy. Nevertheless, the effectiveness of sorafenib is greatly restricted due to resistance. Research has shown that exosomes and circular RNAs play a vital role in the cancer's malignant advancement. However, the significance of exosomal circular RNAs in the development of resistance to sorafenib in HCC remains uncertain. METHODS: Ultracentrifugation was utilized to isolate exosomes (Exo-SR) from the sorafenib-resistant HCC cells' culture medium. Transcriptome sequencing and differential expression gene analysis were used to identify the targets of Exo-SR action in HCC cells. To identify the targets of Exo-SR action in HCC cells, transcriptome sequencing and analysis of differential expression genes were employed. To evaluate the impact of exosomal circUPF2 on resistance to sorafenib in HCC, experiments involving gain-of-function and loss-of-function were conducted. RNA pull-down assays and mass spectrometry analysis were performed to identify the RNA-binding proteins interacting with circUPF2. RNA immunoprecipitation (RIP), RNA pull-down, electrophoretic mobility shift assay (EMSA), immunofluorescence (IF) -fluorescence in situ hybridization (FISH), and rescue assays were used to validate the interactions among circUPF2, IGF2BP2 and SLC7A11. Finally, a tumor xenograft assay was used to examine the biological functions and underlying mechanisms of Exo-SR and circUPF2 in vivo. RESULTS: A novel exosomal circRNA, circUPF2, was identified and revealed to be significantly enriched in Exo-SR. Exosomes with enriched circUPF2 enhanced sorafenib resistance by promoting SLC7A11 expression and suppressing ferroptosis in HCC cells. Mechanistically, circUPF2 acts as a framework to enhance the creation of the circUPF2-IGF2BP2-SLC7A11 ternary complex contributing to the stabilization of SLC7A11 mRNA. Consequently, exosomal circUPF2 promotes SLC7A11 expression and enhances the function of system Xc- in HCC cells, leading to decreased sensitivity to ferroptosis and resistance to sorafenib. CONCLUSIONS: The resistance to sorafenib in HCC is facilitated by the exosomal circUPF2, which promotes the formation of the circUPF2-IGF2BP2-SLC7A11 ternary complex and increases the stability of SLC7A11 mRNA. Focusing on exosomal circUPF2 could potentially be an innovative approach for HCC treatment.

Hsa_circ_0051908 Promotes Hepatocellular Carcinoma Progression by Regulating the Epithelial-Mesenchymal Transition Process.

Materials and Methods: Hsa_circ_0051908 expression was determined using RT-qPCR. HCC cell proliferation, apoptosis, invasion, and migration were assessed using CCK-8 assay, EdU staining, TUNEL staining, flow cytometry, and transwell assay. The molecular mechanism was analyzed using western blotting. In addition, the role of hsa_circ_0051908 in tumor growth was evaluated in vivo. Results: Hsa_circ_0051908 expression was increased in both HCC tissues and cell lines. The proliferation, migration, and invasion of HCC cells were significantly decreased after hsa_circ_0051908 knockdown, while cell apoptosis was notably increased. Furthermore, we found that hsa_circ_0051908 silencing downregulated vimentin and Snail and upregulated E-cadherin. In vivo, hsa_circ_0051908 silencing significantly inhibited the growth of the tumor. Conclusions: Our data provide evidence that hsa_circ_0051908 promotes HCC progression partially by mediating the epithelial-mesenchymal transition process, and it may be used for HCC treatment.

Exosomes from hypoxic ADSCs ameliorate neuronal damage post spinal cord injury through circ-Wdfy3 delivery and inhibition of ferroptosis.

BACKGROUND: Exosomes generated from adipose-derived mesenchymal stem cells (Exos), and in particular hypoxia-pretreated ADSCs (HExos), possess therapeutic properties that promote spinal cord repair following spinal cord injury (SCI). Nevertheless, the regulatory mechanisms through which HExos exert their effects remain unclear. METHODS: Here, next-generation sequencing (NGS) was utilized to examine abnormal circRNA expression comparing HExos to Exos. Bioinformatics analysis and RNA pulldown assays together with luciferase reporter assays were applied to determine interactions among miRNAs, mRNAs and circRNAs. ELISA and immunofluorescence staining were used to examine inflammatory cytokine levels, apoptosis and ROS deposition in LPS-treated HT-22 cells, respectively. The therapeutic effects of Exos and HExos on a mouse model of SCI were analyzed by immunohistochemistry and immunofluorescence staining. RESULTS: Our findings confirmed that HExos have more significant therapeutic influences on decreasing ROS and inflammatory cytokine levels post-SCI than Exos. NGS revealed that circ-Wdfy3 expression levels were significantly higher in HExos than Exos. Downregulation of circ-Wdfy3 led to a decrease in HExo-induced therapeutic effects on spinal cord repair post-SCI, indicating that circ-Wdfy3 has a critical role in the regulation of HExo-mediated protection against SCI. Our bioinformatics, RNA pulldown and luciferase reporter data demonstrated that GPX4 and miR-423-3p were downstream targets of circ-Wdfy3. GPX4 downregulation or miR-423-3p overexpression reversed the protective effects of circ-Wdfy3 on LPS-treated HT-22 cells. Furthermore, overexpression of circ-Wdfy3 led to an in increase in the Exo-induced therapeutic effects on spinal cord repair post-SCI through the inhibition of ferroptosis. CONCLUSIONS: circ-WDfy3-overexpressing Exos promote spinal cord repair post-SCI through mediation of ferroptosis via the miR-138-5p/GPX4 pathway.

Transcriptome- and proteome-wide effects of a circular RNA encompassing four early exons of the spinal muscular atrophy genes.

Spinal muscular atrophy (SMA) genes, SMN1 and SMN2 (hereinafter referred to as SMN1/2), produce multiple circular RNAs (circRNAs), including C2A-2B-3-4 that encompasses early exons 2A, 2B, 3 and 4. C2A-2B-3-4 is a universally and abundantly expressed circRNA of SMN1/2. Here we report the transcriptome- and proteome-wide effects of overexpression of C2A-2B-3-4 in inducible HEK293 cells. Our RNA-Seq analysis revealed altered expression of ~ 15% genes (4172 genes) by C2A-2B-3-4. About half of the affected genes by C2A-2B-3-4 remained unaffected by L2A-2B-3-4, a linear transcript encompassing exons 2A, 2B, 3 and 4 of SMN1/2. These findings underscore the unique role of the structural context of C2A-2B-3-4 in gene regulation. A surprisingly high number of upregulated genes by C2A-2B-3-4 were located on chromosomes 4 and 7, whereas many of the downregulated genes were located on chromosomes 10 and X. Supporting a cross-regulation of SMN1/2 transcripts, C2A-2B-3-4 and L2A-2B-3-4 upregulated and downregulated SMN1/2 mRNAs, respectively. Proteome analysis revealed 61 upregulated and 57 downregulated proteins by C2A-2B-3-4 with very limited overlap with those affected by L2A-2B-3-4. Independent validations confirmed the effect of C2A-2B-3-4 on expression of genes associated with chromatin remodeling, transcription, spliceosome function, ribosome biogenesis, lipid metabolism, cytoskeletal formation, cell proliferation and neuromuscular junction formation. Our findings reveal a broad role of C2A-2B-3-4, and expands our understanding of functions of SMN1/2 genes.

Subcellular localization of circular RNAs: Where and why.

Localization of RNAs at specific subcellular locations regulating various local cellular events has gained much attention recently. Like most other classes of RNAs, the function of newly discovered circular RNAs (circRNAs) is predominantly determined by their association with different cellular factors in the cell. CircRNAs function as transcriptional and posttranscriptional regulators of gene expression by interacting with transcription factors, splicing regulators, RNA-binding proteins, and microRNAs or by translating into functional polypeptides. Hence, studying their subcellular localization to assess their function is essential. The discovery of more than a million circRNA and increasing evidence of their involvement in development and diseases require a thorough analysis of their subcellular localization linking to their biological functions. Here, we summarize current knowledge of circRNA localization in cells and extracellular vesicles, factors regulating their subcellular localization, and the implications of circRNA localization on their cellular functions. Given the discovery of many circRNAs in all life forms and their implications in pathophysiology, we discuss the challenges in studying circRNA localization and the opportunities for unlocking the mystery of circRNA functions.

Exosome-transmitted circular RNA circ-LMO7 facilitates the progression of osteosarcoma by regulating miR-21-5p/ARHGAP24 axis.

The potential function and mechanism of circRNAs in regulating malignant performances of Osteosarcoma (OS) cells have not been well investigated. The expression level of CircLMO7, miR-21-5p and ARHGAP24 were detected by RT-qPCR. The relationship between miR-21-5p and circ-LMO7, as well as between miR-21-5p and ARHGAP24, was predicted and examined through bioinformatics analysis and luciferase reporter gene experiments. Moreover, OS cell growth, invasion, migration, and apoptosis were detected using the cell counting kit-8 (CCK-8), transwell and flow cytometry assays, respectively. ARHGAP24 protein level was measured using western blotting. In present study, we choose to investigate the role and mechanism of circ-LOM7 on OS cell proliferation, migration and invasion. circ-LOM7 was found to be down-regulated in OS tissues and cell lines. Enforced expression of circ-LOM7 suppressed the growth, invasion, and migration of OS cells. In contrast, decreasing circ-LMO7 expression had opposite effects. Furthermore, miR-21-5p was predicted to be sponged by circ-LMO7, and had an opposite role of circ-LMO7 in OS. Moreover, ARHGAP24 served as miR-21-5p's downstream target. Mechanistically, circ-LMO7 was packed in exosomes and acted as a cancer-suppresser on OS by sponging miR-21-5p and upregulating the expression of ARHGAP24. The exosomal circ-LMO7 expression was significantly decreased in OS cell exosomes, and co-culture experiments showed that exosomal circ-LMO7 suppressed the proliferation ability of OS cells. Circ-LMO7 exerts as a tumor suppressor in OS, and the circ-LMO7/miR-21-5P/ARHGAP24 axis is involved in OS progression.