Exosomal circRNA Research Articles

Comprehensive analysis of exosomal circRNA-miRNA-mRNA network in breast cancer.

Exosomal circRNAs played critical roles in tumor development and progression and might be novel biomarkers in the diagnosis and treatment of various cancers. However, the biological functions and clinical implications of exosomal circRNAs in breast cancer are unclear. Expression profiles of exosomal circRNAs in exoRBase 2.0 database were used to identify differentially expressed exosomal circRNAs in breast cancer. The LASSO and SVM-RFE algorithm followed by multivariate logistic regression analysis were performed to construct the diagnostic model. The target genes of circRNAs were selected by combing differential expression analysis and CSCD, TargetScan and ENCORI database. Univariate and multivariate survival analysis were conducted to construct a survival-associated exosomal circRNA-miRNA-mRNA network. GSVA and CIBERSORT algorithm were used to evaluate the cancer hallmarks and immune cells in breast cancer and spearman correlation analysis was used to investigate their correlations with the circRNA-miRNA-mRNA network. A total of 347 upregulated and 3 downregulated exosomal circRNAs were identified in breast cancer patients. The diagnostic model basing on 14 exosomal circRNAs showed a high area under the curve (AUC) value in both the training (AUC = 0.98) and validation dataset (AUC = 0.94). A total of 70 miRNAs and 1147 mRNAs were selected as the downstream targets of circRNAs and were revealed to participate into tumor-associated pathways including PI3K-AKT, MAPK, RAS and RAP1 pathways, calcium signaling pathways and transcriptional misregulations. The constructed survival-associated exosomal circRNA-miRNA-mRNA network contained 9 exosomal circRNAs, 12 miRNAs and 10 mRNAs and showed complicated correlations and interactions within networks. Cancer hallmark pathways including TGF-β, KRAS and MYC signaling pathways, tumor angiogenesis, epithelial-mesenchymal transition, DNA repair and G2M checkpoint and immune cells including CD4+ and CD8+ T cells, dendritic cells, mast cells, macrophage cells, memory B cells and natural killer cells were closely correlated with the circRNA-miRNA-mRNA network. Our study was the first to systematically analyze the exosomal circRNAs in breast cancer. We established an exosomal circRNA diagnostic model and constructed a survival-associated exosomal circRNA-miRNA-mRNA network. Our results revealed the complicated functions and potential mechanisms of exosomal circRNA-miRNA-mRNA network in breast cancer which need to be further validated in the future.

Comprehensive analysis of exosomal circRNA, lncRNA, and mRNA profiles to identify the potential RNAs involved in the pathogenesis of venous malformation.

Venous malformation (VM) is a kind of congenital vascular anomaly with a high incidence of recurrence, detailed pathogenesis and standard treatment of VM still lack now. Increasing evidence showed exosomal RNA plays a pivotal role in various diseases. However, the underlying mechanism of VM based on the potential differentially exosomal RNAs remains unclear. Comparative high-throughput sequencing with serum exosomes from three VM patients and three healthy donors was used to explore differentially expressed (DE) circRNAs, DE lncRNAs, and DE mRNAs involving the formation of VM. We identified and verified DE circRNAs, DE lncRNAs, and DE mRNAs via qRT-PCR assay. We explored the potential functions of these exosomal DE non-coding RNAs via performing further Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis. Besides, circRNA/lncRNA-miRNA-mRNA linkages were also constructed to find their potential relationships in VM. A total of 121 circRNAs, 53 lncRNAs, and 42 mRNAs (|log2 FC| ≥ 2.0, FDR <0.05, n=3) were determined to be differentially expressed. QRT-PCR validated that these top-changed DE circRNAs, lncRNAs, and mRNAs had significant expression changes. Functional studies demonstrated that DE circRNAs play a pivotal role in thyroid hormone signaling pathway, DE lncRNAs function as a key regulator in MAPK signaling pathway and DE miRNAs participate in the process of hepatocellular carcinoma mostly. Our study comprehensively depicted exosomal DE non-coding RNAs networks related to the pathogenesis of VM which can provide new insight into, a novel target for treating VM.

BMSC-Derived Exosomal CircHIPK3 Promotes Osteogenic Differentiation of MC3T3-E1 Cells via Mitophagy

Exosome-based therapy is emerging as a promising strategy to promote bone regeneration due to exosomal bioactive cargos, among which circular RNA (circRNA) has recently been recognized as the key effector. The role of exosomal circRNA derived from bone marrow mesenchymal stem cells (BMSCs) has not been well-defined. The present study aimed to clarify the regulatory function and molecular mechanism of BMSC-derived exosomal circRNA in osteogenesis. Exosomes derived from bone marrow mesenchymal stem cells (BMSC-Exos) were isolated and identified. BMSC-Exos' pro-osteogenic effect on MC3T3-E1 cells was validated by alkaline phosphatase (ALP) activity and Alizarin Red staining. Through bioinformatic analysis and molecular experiments, circHIPK3 was selected and verified as the key circRNA of BMSC-Exos to promote osteoblast differentiation of MC3T3-E1 cells. Mechanistically, circHIPK3 acted as an miR-29a-5p sponge and functioned in mitophagy via targeting miR-29a-5p and PINK1. Additionally, we showed that the mitophagy level of MC3T3-E1 cells were mediated by BMSC-Exos, which promoted the osteogenic differentiation. Collectively, our results revealed an important role for BMSC-derived exosomal circHIPK3 in osteogenesis. These findings provide a potentially effective therapeutic strategy for bone regeneration.

The aggravate role of exosomal circRNA11:120406118|12040782 on macrophage pyroptosis through miR-30b-5p/NLRP3 axis in silica-induced lung fibrosis.

Silica dust inhalation could lead to silicosis, and there is no specific biomarker for its early diagnosis and no effective treatment due to the lack of research on its pathogenesis. The homeostasis of macrophages was considered to be crucial during the development of silicosis from persistent chronic inflammation to irreversible fibrosis. However, its regulatory mechanism and the communication between macrophages and others are still not clear. Exosomal circRNAs emerge as favorable candidates for cellular communication. Therefore, our study aimed to illustrate the regulatory mechanism of silicosis from the view of exosomal circRNAs. Our study identified a novel exosomal circRNA, circRNA11:120406118|12040782, in the peripheral serum of silicosis patients. Furthermore, the detailed role of circRNA11:120406118|12040782 was investigated both in silicosis mouse model and in silica-stimulated macrophages and fibroblasts. On the one hand, circRNA11:120406118|12040782 was shown to regulate silica-stimulated macrophage pyroptosis through circRNA11:120406118|12040782/miR-30b-5p/NLRP3 network. And this macrophage-derived cirRNA could promote the activation of fibroblasts. On the other hand, overexpressing miR-30b-5p, the crucial component of circRNA11:120406118|12040782/miR-30b-5p/NLRP3 regulatory network, could inhibit pyroptosis and attenuate silica-induced lung inflammation and fibrosis in mice. Our findings suggested that exosomal circRNA11:120406118|12040782 could aggravate NLRP3-mediated macrophages pyroptosis through sponging miR-30b-5p in silicosis development, which provide an experimental basis and shed light on the early diagnosis and treatment of silicosis.

Developing and validating a survival prediction model based on blood exosomal ceRNA network in patients with PAAD

BackgroundAmong the most lethal cancers, pancreatic adenocarcinoma (PAAD) is an essential component of digestive system malignancies that still lacks effective diagnosis and treatment methods. As exosomes and competing endogenous RNA (ceRNA) regulatory networks in tumors go deeper, we expect to construct a ceRNA regulatory network derived from blood exosomes of PAAD patients by bioinformatics methods and develop a survival prediction model based on it.MethodsBlood exosome sequencing data of PAAD patients and normal controls were downloaded from the exoRbase database, and the expression profiles of exosomal mRNA, lncRNA, and circRNA were differentially analyzed by R. The related mRNA, circRNA, lncRNA, and their corresponding miRNA prediction data were imported into Cytoscape software to visualize the ceRNA network. Then, we conducted GO and KEGG enrichment analysis of mRNA in the ceRNA network. Genes that express differently in pancreatic cancer tissues compared with normal tissues and associate with survival (P < 0.05) were determined as Hub genes by GEPIA. We identified optimal prognosis-related differentially expressed mRNAs (DEmRNAs) and generated a risk score model by performing univariate and multivariate Cox regression analyses.Results205 DEmRNAs, 118 differentially expressed lncRNAs (DElncRNAs), and 98 differentially expressed circRNAs (DEcircRNAs) were screened out. We constructed the ceRNA network, and a total of 26 mRNA nodes, 7 lncRNA nodes, 6 circRNA nodes, and 16 miRNA nodes were identified. KEGG enrichment analysis showed that the DEmRNAs in the regulatory network were mainly enriched in Human papillomavirus infection, PI3K-Akt signaling pathway, Osteoclast differentiation, and ECM-receptor interaction. Next, six hub genes (S100A14, KRT8, KRT19, MAL2, MYO5B, PSCA) were determined through GEPIA. They all showed significantly increased expression in cancer tissues compared with control groups, and their high expression pointed to adverse survival. Two optimal prognostic-related DEmRNAs, MYO5B (HR = 1.41, P < 0.05) and PSCA (HR = 1.10, P < 0.05) were included to construct the survival prediction model.ConclusionIn this study, we successfully constructed a ceRNA regulatory network in blood exosomes from PAAD patients and developed a two-gene survival prediction model that provided new targets which shall aid in diagnosing and treating PAAD.

Gastric cancer stem cell-derived exosomes promoted tobacco smoke-triggered development of gastric cancer by inducing the expression of circ670.

As one of the most common malignant cancers in the world, gastric cancer is caused by mang factors among which tobacco smoke is an important risk factor. Gastric cancer stem cells (GCSCs) and the derived exosomes play a key role in the occurrence and development of gastric cancer, and exosomal circRNA is considered as a new regulatory factor in the development of gastric cancer. However, it is unclear whether tobacco smoke can affect exosomes and their transport circRNAs to promote the development of gastric cancer. Herein, we provided a new insight into tobacco smoke promoting the progression of gastric cancer. In the present study, we demonstrated that tobacco smoke-induced exosomes promoted the spheroidizing ability, stemness genes expression, and epithelial-mesenchymal transition (EMT) process of GCSCs. We further found that hsa-circRNA-000670 (circ670) was up-regulated in tissues of gastric cancer patients with smoking history, tobacco smoke-induced GCSCs, and their exosomes. Functional assays have shown that circ670 knockdown inhibited the stemness and EMT process of GCSCs, whereas circ670 overexpression appeared to have an opposite effect. Our findings indicated that exosomal circ670 promotes the development of tobacco smoke-induced gastric cancer, which may provide insight into the mechanism of tobacco smoke promoting the progression of gastric cancer.

Exosomal circNFIX promotes angiogenesis in ovarian cancer via miR-518a-3p/TRIM44 axis.

Ovarian cancer (OC) is a gynecological cancer with high mortality. OC-derived exosomal circRNAs can regulate angiogenesis. This study aims to explore the role and mechanism of exosomal circRNA nuclear factor I X (CircNFIX) derived from OC cells in angiogenesis. Quantitative real-time polymerase chain reaction was employed to evaluate the levels of circNFIX, miR-518a-3p, and tripartite motif protein 44 (TRIM44) in OC and adjacent tissues. Exosomes from the ovarian surface epithelial cell (HOSEpiC) and OC cells (SKOV3 or OVCAR3) were isolated by differential centrifugation. Exosomes were cocultured with the human umbilical vein endothelial cells (HUVECs). The angiogenesis capacity was analyzed by Tube formation assay. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and Transwell assays were used to determine the cell viability and migration ability. The dual-luciferase report, RNA immunoprecipitation (RIP), and RNA pull-down assays were applied to validate the gene's interaction. CircNFIX and TRIM44 expression were higher and miR-518a-3p was lower in OC tissues than in the adjacent tissues. Upregulated circNFIX and TRIM44 were significantly correlated with the tumor size and International Federation of Gynecology and Obstetrics (FIGO) stage of OC patients. HUVECs treated OC-derived exosomes had higher proliferation, migration, and angiogenesis capacities than the control group. While OC-derived exosomal circNFIX silencing restrained HUVECs' proliferation, migration, and angiogenesis, compared with the OC-derived exosomes group. OC-derived exosomal circNFIX positively regulated TRIM44 expression by targeting miR-518a-3p in HUVECs. OC-derived exosomal circNFIX promoted angiogenesis by regulating the Janus-activated kinase/signal transducer and activator of transcription 1 (JAK/STAT1) pathway via miR-518a-3p/TRIM44 axis in HUVECs.

Construction of a ceRNA network in polycystic ovary syndrome (PCOS) driven by exosomal lncRNA.

Polycystic ovary syndrome (PCOS), a common and frustrating syndrome in women of reproductive age, is characterized by symptoms including hyperandrogenemia, ovulation dysfunction, and polycystic ovaries. The role of competitive endogenous RNA (ceRNA) networks is receiving increasing attention and has been reported in multiple complicated diseases, such as various carcinomas, endometriosis, and tubal factor infertility. However, the association of ceRNA networks with the pathogenesis of PCOS remains unclear. This study aimed to construct a ceRNA network orchestrated by exosomal lnRNA and circRNA in PCOS. We screened RNA data of 34 samples from the Gene Expression Omnibus (GEO) database for differentially expressed lncRNAs (DELs), miRNAs (DEMs), mRNAs (DEGs), and circRNA associated with the progression of PCOS (PCOS, n = 17 vs. normal, n = 17). A protein-protein interaction (PPI) network, gene set enrichment analysis (GSEA), and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted. Importantly, the function of the ceRNA network was explored using GO and KEGG enrichment analyses. We identified 46 DELs (25 upregulated and 21 downregulated), 31 DEMs (20 upregulated and 11 downregulated), 165 DEGs (52 upregulated and 113 downregulated), and 1 differentially expressed circRNA. The PPI network had 79 nodes and 112 edges. The GSEA results showed that these genes were mainly related to oxidative phosphorylation; TNF signaling pathways; and valine, leucine, and isoleucine degradation. GO and KEGG analyses revealed that the DEGs were significantly enriched in lipid metabolism, peroxisome proliferator-activated receptor (PPAR) signaling pathways, and fatty acid metabolism. Additionally, we constructed a novel PCOS-associated lncRNA-miRNA-mRNA ceRNA triple network and a circRNA-related network. Thereafter, we described the potential roles played by follicular fluid exosomes in PCOS. Our present study describes the molecular pathogenesis of PCOS in human ovarian granulosa cells at the post-transcriptional level, which provides new insights for the clinical diagnosis and treatment of PCOS and further scientific research.

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.

Prediction of COPD acute exacerbation in response to air pollution using exosomal circRNA profile and Machine learning.

Ambient fine particulate matter (PM2.5) is linked to an increased risk of chronic obstructive pulmonary disease (COPD) exacerbations, which significantly increase the risk of mortality in COPD patients. Identifying the subtype of COPD patients who are sensitive to environmental aggressions is necessary. Using in vitro and in vivo PM2.5 exposure models, we demonstrate that exosomal hsa_circ_0005045 is upregulated by PM2.5 and binds to the protein cargo peroxiredoxin2, which functionally aggravates hallmarks of COPD by recruiting neutrophil elastase and triggering in situ release of tumor necrosis factor (TNF)-α by inflammatory cells. The biological function of hsa_circ_0005045 associated with aggravation of COPD is validated using exosome-transplantation and conditional circRNA-knockdown murine models. By sorting the major components of PM2.5, we find that PM2.5-bound heavy metals, which are distinguishable from the components of cigarette smoke, trigger the elevation of exosomal hsa_circ_0005045. Finally, using machine learning models in a cohort with 327 COPD patients, the PM2.5 exposure-sensitive COPD patients are characterized by relatively high hsa_circ_0005045 expression, non-smoking, and group C (mMRC 0-1 (or CAT<10) and≥2 exacerbations (or≥1 exacerbation leading to hospital admission) in the past year). Thus, our results suggest that environmental reduction in PM2.5 emission provides a targeted approach to protecting non-smoking COPD patients against air pollution-related disease exacerbation.

Exosomal circRNA Scm-like with four malignant brain tumor domains 2 (circ-SFMBT2) enhances the docetaxel resistance of prostate cancer via the microRNA-136-5p/tribbles homolog 1 pathway.

Exosomal circular RNA was found to mediate cancer chemoresistance. However, whether exosomal circRNA Scm-like with four malignant brain tumor domains 2 (circ-SFMBT2) was involved in the chemoresistance of prostate cancer (PCa) remains unclear. The docetaxel (DTX) resistance of PCa cells was analyzed by Cell Counting Kit 8 assay. Quantitative real-time PCR was used to measure circSFMBT2, microRNA (miR)-136-5p and tribbles homolog 1 (TRIB1) expression. Cell proliferation, apoptosis, migration and invasion were analyzed by 5-ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry, wound-healing assay and transwell assay. RNA interaction was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Protein expression was measured by western blot analysis. Exosomes-extracted from cells were identified by transmission electron microscope, nanoparticles tracking analysis and western blot. Xenograft mice models were constructed to analyze the effect of exosomal circSFMBT2 on the DTX sensitivity of PCa tumors in vivo. CircSFMBT2 was upregulated in DTX-resistant PCa cells, and its knockdown enhanced the DTX sensitivity of DTX-resistant PCa cells by suppressing cell proliferation, migration, invasion and enhancing apoptosis. CircSFMBT2 severed as miR-136-5p sponge to positively regulate TRIB1. The regulation of circSFMBT2 knockdown on the DTX sensitivity of DTX-resistant PCa cells could be reversed by miR-136-5p inhibitor or TRIB1 overexpression. Exosomal circSFMBT2 from DTX-resistant PCa could increase the DTX resistance of normal PCa cells. In addition, exosomal circSFMBT2 also enhanced the DTX resistance of PCa tumors in vivo, and it was highly expressed in the serum of DTX-resistance PCa patients. Exosomal circSFMBT2 enhanced the DTX resistance of PCa by miR-136-5p/TRIB1 axis, indicating that circSFMBT2 might be a potential target for the treatment of PCa chemoresistance.

EVALUATION OF EXOSOME-DERIVED CircRNA EXPRESSION PATTERNS IN LUNG CANCER RADIOTHERAPY

Background: Radiotherapy is an effective method used in the treatment of lung cancer. However, it is very difficult to assess the clinical manifestations of radiotherapy at an early stage and the relevant criteria are very limited. Exosome-derived circRNAs (exo-circRNAs) have potential as biomarkers for early a cancer diagnosis and treatment assessment as exosomes are present in a variety of body fluids and circRNAs in exosomes exhibit high stability. Therefore, we aimed to investigate for potential biomarker candidates by evaluating changes in exo-circRNA expression with radiotherapy in lung cancer. Method: In this study, lung cancer epithelial (A549) and healthy lung fibroblast (WI38) cell lines were used. After colony assay, both cell lines were irradiated with 4 Gy and exosomes were isolated by ultracentrifugation method. Exosomes were characterized by flow cytometry and nanoparticle tracking analysis, and RNA isolation was performed by trizol protocol. Then, the expression levels of 40 exo-circRNAs in all groups were examined by RT-qPCR. Results: Of the exosomal circRNA investigated, 5 exo-circRNAs were found to be expressed in at least one of the groups, including A549, WI38, irradiated A549 and irradiated WI-38 cell lines. In particular, hsa_circ_0014235 and hsa_circ_0001900, which were found to be expressed in all groups, were more upregulated in cancer cell lines than healthy cell line. Significant changes were detected in the expression levels of these exo-circRNAs after irradiation. Conclusions: Differential expression of exo-circRNAs between cancer and healthy group and alterations by irradiation suggests that they may be potential diagnostic and monitoring biomarkers in cancer.

Identification of exosomal circRNA CD226 as a potent driver of nonsmall cell lung cancer through miR-1224-3p/high mobility group AT-hook 2 axis.

Circular RNAs (circRNAs) are crucial for the pathogenesis of nonsmall lung cancer (NSCLC). Here, we set out to unravel the precise function of circRNA CD226 (circCD226) in NSCLC pathogenesis. The exosomes from serum specimens were observed by transmission electron microscopy. CircCD226, miR-1224-3p and high mobility group AT-hook 2 (HMGA2) were quantified by qRT-PCR, western blot and immunohistochemistry. Actinomycin D and Ribonuclease (RNase) R treatments and subcellular localization assay were used for circCD226 characterization. Cell viability, proliferation, migration, invasion and sphere formation abilities were gauged by CCK-8, EDU, wound-healing, transwell and sphere formation assays, respectively. Directed relationships among circCD226, miR-1224-3p and HMGA2 were examined by RNA pull-down, dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. The abundance of circCD226 was elevated in serum exosomes, tissues and cells of NSCLC. NSCLC serum exosomes enhanced NSCLC cell proliferation, migration, invasion and stemness. Loss of circCD226 impeded cell proliferation, migration, invasion and stemness in vitro , as well as tumor growth in vivo . Mechanistically, circCD226 sponged miR-1224-3p, and miR-1224-3p targeted HMGA2. CircCD226 involved the posttranscriptional regulation of HMGA2 through miR-1224-3p. Moreover, the miR-1224-3p/HMGA2 axis was identified as a functionally downstream effector of circCD226 in regulating NSCLC cell behaviors. Our study identifies circCD226 as a potential driver in NSCLC development depending on the regulation of miR-1224-3p/HMGA2 axis.

Serum and Serum Exosomal CircRNAs hsa_circ_0001492, hsa_circ_0001439, and hsa_circ_0000896 as Diagnostic Biomarkers for Lung Adenocarcinoma.

BackgroundCircular RNAs (circRNAs) play an important role in tumorigenesis and several circulating circRNA signatures are closely associated with tumor diagnosis. However, the expression and clinical significance of the two forms of circulating circRNAs, serum and serum exosomal, in patients with lung adenocarcinoma (LUAD), have not been characterized.MethodsThree differentially expressed exosomal circRNAs, hsa_circ_0001492, hsa_circ_0001439, and hsa_circ_0000896, were selected based on previous exosomal circRNA sequencing data analyses of LUAD patients. The expression of these circRNAs in serum and serum-derived exosomes of LUAD patients was assessed using quantitative real-time PCR (qRT-PCR), and correlations between circRNA expression and clinicopathological characteristics were analyzed. The reliability of serum and serum exosomal hsa_circ_0001492, hsa_circ_0001439, and hsa_circ_0000896 to diagnose LUAD was evaluated using receiver operating characteristic (ROC) analysis.ResultsExpression of serum and serum exosomal hsa_circ_0001492, hsa_circ_0001439, and hsa_circ_0000896 were significantly higher in LUAD patients than in healthy donors, and significantly lower after surgery. These three serum exosomal circRNAs were also associated with a higher cancer stage. Exosomal hsa_circ_0001492 expression was positively correlated with carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE) levels. An association between the expression of the three serum circRNAs and clinical characteristics was not observed. In addition, the three serum exosomal circRNAs had higher diagnostic sensitivity and specificity than the serum circRNAs, and the area under the curve (AUC) of all three serum exosomal circRNAs was >0.75. The combination of exosomal hsa_circ_0001492, hsa_circ_0001439, and hsa_circ_0000896 had better diagnostic sensitivity and specificity than that of a single marker, with an AUC value of 0.805.ConclusionsThe serum and serum exosomal circRNAs, hsa_circ_0001492, hsa_circ_0001439, and hsa_circ_0000896, were upregulated in LUAD patients. Serum exosomal circRNAs may serve as more effective biomarkers than serum circRNAs for LUAD diagnosis and may further aid the detection of this disease.

CircRNA has_circ_0069313 induced OSCC immunity escape by miR-325-3p-Foxp3 axes in both OSCC cells and Treg cells.

Introduction: CircRNAs are engaged in the tumorigenesis and progression of oral squamous cancer cells (OSCC). However, the function and underlying mechanism of circRNAs on tumor-associated immunity escape are largely unknown.Materials and methods: We analyzed the expression pattern of has_circ_0069313 in our in-house database and its correlation with OSCC prognosis. Immunohistochemistry was applied to detected PDL1 expression. RNA fluorescence in situ hybridization was applied to detect subcellular location of circRNA. A luciferase activity assay was used to detect the interaction of has_circ_0069313 and miR-325-3p and its downstream target, Foxp3. Exosomes were collected to detect the exosomal circRNAs and co-culture assays were performed to detect the function of exosomal circRNAs on Tregs.Results: has_circ_0069313 was upregulated in OSCC tissues and predicts poor prognosis. has_circ_0069313 promotes immunity escape through inhibiting miR-325-3p-induced Foxp3 degradation. has_circ_0069313 is an exosomal circRNA and the transfer of has_circ_0069313 to Treg cells promotes the Treg function through maintaining Foxp3 levels.Conclusion: Our results indicate that has_circ_0069313 induces OSCC immunity escape via the miR-325-3p-Foxp3 axis in both OSCC cells and Treg cells.

Exosomal Circsafb2 Reshaping Tumor Environment to Promote Renal Cell Carcinoma Progression by Mediating M2 Macrophage Polarization.

BackgroundMacrophages are the most abundant infiltrating immune-related stromal cells present in and around tumors, showing different phenotypes and functions. M2 macrophages mainly exert immunosuppressive functions and promote tumor growth. Exosomes are emerging as important mediators of cross-talk between tumor cells and the microenvironment. CircRNAs are novel members of non-coding RNAs that regulate cancer proliferation and progression. However, the mechanism by which exosomal circRNA regulates macrophage polarization in renal cell carcinoma (RCC) is still largely unknown.MethodsRCC-derived exosomes were characterized using transmission electron microscopy and nanoparticle tracking analysis (NTA). CCK-8, wound healing, and Transwell assays were performed to assess whether exosomes would affect the proliferation, migration, and invasion of RCC. Furthermore, we performed a bioinformatics analysis to identify circRNAs in RCC serum-derived exosomes from the GEO database. The fluorescence in situ hybridization (FISH) assay was used to detect the cellular distribution of circSAFB2. Bioinformatics analyses (StarBase 2.0) were used to pool the miRNA targets of circSAFB2. Luciferase assays were performed to verify the direct interactions. Western blotting was used to detect markers of macrophage M2 polarization. Lastly, mouse xenograft and bioluminescence imaging were used to examine the clinical relevance of exosomal circSAFB2 in vivo.ResultsWe report the circRNA derived from SAFB2 and evaluate its biological function in promoting the immune escape of RCC. We found that circSAFB2 was highly expressed in RCC tissues and RCC-derived exosomes. Furthermore, we demonstrated that exosomal circSAFB2 mediates the polarization of M2 macrophages through the miR-620/JAK1/STAT3 axis to promote RCC metastasis.ConclusionsOur data first demonstrated that circSAFB2 leads to immune escape from RCC by mediating M2 macrophage polarization via the miR-620/JAK1/STAT3 axis. These findings indicate a novel molecular mechanism of exosomal circSAFB2 in the progression of RCC and implicate circSAFB2 as a target for exosome-mediated tumor immune evasion.

Exosomal circRNA FNDC3B promotes the progression of esophageal squamous cell carcinoma by sponging miR-490-5p and regulating thioredoxin reductase 1 expression

ABSTRACT Exosomal circular RNAs (circRNAs) have been reported to play critical roles in esophageal squamous cell carcinoma (ESCC). We aimed to investigate the function of exosomal circRNA FNDC3B (circFNDC3B). The RNA levels and protein levels were examined using RT-qPCR and western blot (WB) assays. Colony formation and EdU assays were used to assess cell proliferative ability. Cell migratory and invasive abilities were detected by wound healing and transwell assays. Cell apoptosis was measured by flow cytometry. Glycolysis was measured using commercial kits. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were applied to examine the morphology and size of exosomes. Dual-luciferase reporter, RIP and RNA pull-down assays assessed the interaction of miR-490-5p with circFNDC3B or thioredoxin reductase 1 (TXNRD1). Xenograft tumor model determined the role of exosomal circFNDC3B in vivo. We observed that circFNDC3B was upregulated in ESCC samples and cells, as well as ESCC-derived exosomes. CircFNDC3B could be delivered via exosomes in tumor cells, and the colony formation, proliferation, migration, invasion, glycolysis, and in vivo growth ability of recipient cells were weakened after co-incubation with exosomal circFNDC3B-knockdown donor cells. CircFNDC3B was a miR-490-5p sponge, and miR-490-5p inhibition reversed the role of exosomal circFNDC3B-downregulating in ESCC cells. TXNRD1 was a miR-490-5p target, and TXNRD1 elevation weakened the anti-cancer function of miR-490-5p upregulation in ESCC cells. CircFNDC3B mediated TXNRD1 expression by interacting with miR-490-5p. In conclusion, exosomal circFNDC3B drove ESCC progression via regulating the miR-490-5p/TXNRD1 axis. Abbreviations EC: esophageal cancer; ESCC: esophageal squamous cell carcinoma; circRNA: circular RNA; WB: western blot; TEM: transmission electron microscopy; NTA: nanoparticle tracking analysis; TXNRD1: thioredoxin reductase 1; IHC: immunohistochemistry; RT-qPCR: reverse transcription-polymerase quantitative chain reaction; GLUT1: glucose transport protein type 1; LDHA: lactate dehydrogenase A

Exosomal circRNA BTG2 derived from RBP-J overexpressed-macrophages inhibits glioma progression via miR-25-3p/PTEN

Macrophage-derived exosomes (Mφ-Exos) are involved in tumor progression, but its role in glioma is not fully understood. RBP-J is related to macrophage activation. In this study, we assess the role of exosomes derived from RBP-J-overexpressed macrophages (RBP-J OE Mφ-Exos) in glioma. The circular RNA (circRNA) profiles in RBP-J OE Mφ-Exos and THP-1-like macrophages (WT Mφ)-Exos were evaluated using circRNA microarray. Then the functions of Mφ-Exo-circRNA in glioma cells were assessed via CCK-8, EdU, Transwell invasion, and nude mouse assays. Besides, luciferase reporter assay, RNA immunoprecipitation, and Pearson’s correlation analysis were adopted to confirm interactions. We found that circRNA BTG (circBTG2) is upregulated in RBP-J OE Mφ-Exos compared to WT Mφ-Exos. RBP-J OE Mφ-Exos co-culture and circBTG2 overexpression inhibited proliferation and invasion of glioma cells, whereas circBTG2 knockdown promotes tumor growth in vivo. The effects of RBP-J OE Mφ-Exos on glioma cells can be reversed by the circBTG2 knockdown. In conclusions, Exo-circBTG2 secreted from RBP-J OE Mφ inhibits tumor progression through the circBTG2/miR-25-3p/PTEN pathway, and circBTG2 is probably a diagnostic biomarker and potential target for glioma therapy.

Exosomal circular RNA hsa_circ_0006220, and hsa_circ_0001666 as biomarkers in the diagnosis of pancreatic cancer.

BackgroundPancreatic cancer is a highly malignant tumor of the digestive system.ObjectiveExosomal circular RNA can be used as a biomarker for the early diagnosis of pancreatic cancer.MethodsThe expression of various differentially expressed circRNAs in pancreatic cancer tissues was analyzed by gene chip, exosome expression was verified by electron microscopy and Western blotting, and the expression of exosomal circRNA in pancreatic cancer cells, tissues, and plasma were determined by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR).ResultsCompared with healthy controls, hsa_circ_0006220 and hsa_circ_0001666 were highly expressed in exosomes in the plasma of pancreatic cancer patients. The AUC values were 0.7817 for hsa_circ_0006220, 0.8062 for hsa_circ_0001666, and 0.884 for the combined diagnosis. In addition, clinicopathological features revealed that the expression of hsa_circ_0006220 in plasma exosomes from pancreatic cancer patients was associated with CA19‐9 levels (p = 0.0001) and lymph node metastasis (p = 0.0005). The expression of hsa_circ_0001666 was correlated with both tumor size (p = 0.0157) and CA19‐9 level (p = 0.0001).ConclusionsThe high expression of exosomal hsa_circ_0001666 and hsa_circ_0006220 suggests that these can be used as new biomarkers for the diagnosis and treatment of pancreatic cancer.

Breast cancer derived exosomes promoted angiogenesis of endothelial cells in microenvironment via circHIPK3/miR-124-3p/MTDH axis

Circular RNAs (circRNAs) are important contents in exosomes, which can regulate peripheral cell functions, thus influencing the tumor microenvironment. This work investigated the mechanisms underlying the angiogenesis in peripheral human endothelial cells (ECs) mediated by the breast cancer (BC) cells derived exosomal circRNAs and aimed to explore the biomarkers for the anti-angiogenesis therapy for BC.The BC cell derived exosomes were extracted and the expression level and the circular formation of HIPK3 enclosed was determined. To examine the impact of this exosomal circRNA on ECs, cell viability and tube formation were determined in recipient cells co-cultured with exosomes or transfected with circHIPK3 and the related controls. Target microRNAs (miRNAs) for circHIPK3 and target genes for miRNAs were predicted and confirmed by multiple assays like dual luciferase reporter assay, western blot, and qPCR assays. The existence of the circHIPK3/miR-124-3p/MTDH axis were further confirmed with rescue experiment in mice xenograft model.HIPK3s were mainly in forms of circRNAs and were highly expressed in the BC cell derived exosomes, which could be absorbed by the recipient ECs. The cell viability and angiogenesis in ECs were enhanced when treated with circHIPK3s and decreased when treated with circHIPK3-si. Furthermore, MTDH was proved to be the responsible gene in this process which was regulated by miR-124-3p, the local miRNA sponged by the exosomal circHIPK3.circHIPK3 enclosed in the BC cell-derived exosomes enhanced MTDH expression in the endothelial cell by sponging miR-124-3p, favoring the tube formation in ECs, which might serve as a therapeutic target for anti-angiogenesis therapy for breast cancer.