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Plasma exosomes transfer miR-885-3p targeting AKT/NFκB signaling pathway to improve the sensitivity of intravenous glucocorticoid therapy against Graves ophthalmopathy

ABSTRACT: Graves ophthalmopathy (GO), a manifestation of Graves' disease, is an organ-specific autoimmune disease. Intravenous glucocorticoid therapy (ivGCs) is the first-line treatment for moderate-to-severe and active GO. However, ivGCs is only effective in 70-80% of GO patients. Insensitive patients who choose 12 weeks ivGCs were not only delayed in treatment, but also took the risk of adverse reactions of glucocorticoids. At present, there is still a lack of effective indicators to predict the therapeutic effect of ivGCs. Therefore, the purpose of this study is to find biomarkers that can determine the sensitivity of ivGCs before the formulation of treatment, and to clarify the mechanism of its regulation of ivGCs sensitivity. This study first characterized the miRNA profiles of plasma exosomes by miRNA sequencing to identify miRNAs differentially expressed between GO patients with significant improvement (SI) and non-significant improvement (NSI) after ivGCs treatment. According to the function of the target genes, we screened 10 differentially expressed miRNAs. Results showed that compared with NSI patients, mir-885-3p was up-regulated and mir-4474-3p and mir-615-3p were down regulated in the exosomes of SI patients. Based on statistical difference and miRNA function, mir-885-3p was selected for follow-up study. In vitro functional analysis of exosomes mir-885-3p showed that exosomes from SI patients (SI-exo) could transfer mir-885-3p to orbital fibroblasts (OFs), up-regulate the GRE luciferase reporter gene plasmid activity and the level of glucocorticoid receptor (GR), down-regulate the level of inflammatory factors, and improve the glucocorticoid sensitivity of OFs. In addition, we found that high levels of mir-885-3p could inhibit AKT/NFκB signaling pathway, up‐regulate the GRE plasmid activity and GR level, and down‐regulate the level of inflammatory factors of OFs. Moreover, the improvement of glucocorticoid sensitivity by mir-885-3p transmitted by SI-exo can also be inhibited by AKT/NFκB agonist. Finally, through the in vivo experiment of GO mouse model, we further determined the relationship between exosomes mir‐885‐3p sequence, AKT/NFκB signaling pathway, and glucocorticoid sensitivity. As a conclusion, plasma exosomes deliver mir‐885‐3p and inhibit AKT/NFκB signaling pathway to improve the glucocorticoid sensitivity of OFs. Exosomes mir-885-3p can be used as a biomarker to determine the sensitivity of ivGCs in GO patients.

ORGANISM(S): Homo sapiens

PROVIDER: GSE190515 | GEO | 2022/02/19

REPOSITORIES: GEO

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Project description:Aim: EBV encode at least 44 miRNAs involved in immune regulation and disease progression. Exosomes can be used as carriers of EBV-miRNA-BART intercellular transmission and affect the biological behavior of cells. We characterized exosomes and established a co-culture experiment of exosomes to explore the mechanism of miR-BART1-3p transmission through the exosome pathway and its influence on tumor cell proliferation and invasion. Materials and methods: Exosomes of EBV-positive and EBV-negative gastric cancer cells were characterized by transmission electron microscopy, Nanosight and western blotting, and miRNA expression profiles in exosomes were sequenced with high throughput. Exosomes with high or low expression of miR-BART1-3p were co-cultured with AGS cells to study the effects on proliferation, invasion and migration of gastric cancer cells. The target genes of EBV-miR-BART1-3p were screened and predicted by PITA, miRanda, RNAhybrid, virBase and Diana-Tarbase v.8 databases, and the expression of the target genes after co-culture was detected by qPCR. Results：The exosomes secreted by EBV positive and negative gastric cancer cells range in diameter from 30 nm to 150nm and express the exosomal signature proteins CD63 and CD81. High throughput sequencing showed that exosomes expressed some human miRNAs, among which has-miR-23b-3p, has-miR-320a-3p and has-miR-4521 were highly expressed in AGS-exo. has-miR-21-5p, has-miR-148a-3p and has-miR-7-5p were highly expressed in SNU-719-exo. All EBV miRNAs were expressed in SNU-719 cells and their exosomes, among which EBV- miR-Bart1-5p, EBV- miR-bart22 and EBV- miR-bart16 were the highest in SNU-719 cells. EBV-miR-BART1-5p, EBV-miR-BART10-3p and EBV-miR-BART16 were the highest in SNU-719-exo. After miR-BART1-3p silencing in gastric cancer cells, the proliferation, healing, migration and invasion of tumor cells were significantly improved. Laser confocal microscopy showed that exosomes could carry miRNA into recipient cells. After co-culture with miR-BART1-3p silenced exosomes, the proliferation, healing, migration and invasion of gastric cancer cells were significantly improved. The target gene of miR-BART1-3p was FMA168A, MACC1, CPEB3, ANKRD28 and USP37 after screening by targeted database. CPEB3 was not expressed in all exosome co-cultured cells, while ANKRD28, USP37, MACC1 and FAM168A were all expressed to varying degrees. USP37 and MACC1 are down-regulated after up-regulation of miR-BART1-3p, which may be the key target genes for miR-BART1-3p to regulate the proliferation of gastric cancer cells through exosomes. Conclusions: miR-BART1-3p can affect the growth of tumor cells through exosome pathway. The proliferation, healing, migration and invasion of gastric cancer cells were significantly improved after co-culture with exosomes of miR-BART1-3p silenced expression. USP37 and MACC1 may be potential target genes of miR-BART1-3p in regulating cell proliferation.

Project description:Purpose：Early diagnosis of nasopharyngeal carcinoma (NPC) is vital to improve the prognosis of these patients. However, early diagnosis of NPC is typically challenging.Therefore, we explored the roles and mechanisms of exosomes in plasma of patients with early-stage NPC. Methods: Exosomes in plasma were extracted by ultra-high-speed centrifugation.Western blot and transmission electron microscopy (TEM) were used to verify the purity of exosomes. The sequencing data (6 plasma samples from healthy volunteers vs. 6 NPC plasma samples) were analyzed by principal component analysis (PCA), DESeq2, gene ontology (GO), kyoto encyclopedia of genes and genomes (KEGG), and TargetScan. The differentially expressed miRNAs (DEmiRNAs) were obtained from the dataset (GSE118720) downloaded from the Gene Expression Omnibus (GEO) repository. Additionally, the datasets downloaded from the GEO database (GSE12452, GSE13597, GSE53819, GSE64634) were used to predict the target genes and functions of hsa-miR-1301-3p. qPCR was applied to verify the differences in the expressions of hsa-miR-1301-3p between 10 normal plasma and 10 NPC plasma samples. Results:Western blot, TEM, and Nanoparticle Tracking Analysis showed adequate purity of the extracted exosomes. RNA-seq analysis revealed 21 upregulated miRNAs, and 10 downregulated miRNAs in plasma exosomes of early-stage NPC patients. GO analysis showed that the target genes of DEmiRNAs were mainly enriched in DNA synthesis and transcription regulation. KEGG analysis revealed that DEmiRNAs were mainly enriched in PI3K-Akt and MAPK signaling pathways. Moreover, the expression of hsa-mir-1301-3p was verified to be significantly upregulated in enlarged samples of plasma exosomes. Conclusions:We identified several DEmiRNAs extracted from tumor-derived exosomes between normal plasma and early-stage NPC plasma. Bioinformatics analyses indicated that these DEmiRNAs may be related to NPC development. Our study may provide novel insights into underlying biomarkers and mechanisms of plasma exosomes in early-stage NPC.

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Plasma exosomes transfer miR-885-3p targeting AKT/NFκB signaling pathway to improve the sensitivity of intravenous glucocorticoid therapy against Graves ophthalmopathy

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