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Real-time quantitative PCR analysis of human exosomes

ABSTRACT: Paroxysmal Nocturnal Hemoglobinuria (PNH) is a clonal disease of blood cells caused by the lack of glycosyl phosphatidyl inositol anchored proteins bound to the cell membrane. In consequence, erythrocytes lead to intravascular hemolysis upon complement activation, which promotes high risk of thrombosis, intravascular hemolytic anemia, and bone marrow failure in patients. The mechanisms of thrombosis in PNH are still poorly understood. Treatment with eculizumab reduces intravascular hemolysis and thrombotic risk, but not in all cases. Exosomes are extracellular vesicles released by cells and whose secretion is closely related to the inflammatory status. They participate in cell communication by activating signaling pathways and transferring genetic material and proteins to host cells. In consequence, exosomes may serve as surrogate biomarkers for the prognosis and/or diagnosis of a disease. Isolation of exosomes was carried out from healthy controls and from three groups of PNH patients, i.e. i) with no eculizumab treatment; ii) under treatment with eculizumab that have not suffered thrombosis; and iii) under treatment with eculizumab but that have suffered thrombosis. The miRNAome and proteome was analyzed using plasma focus miRNAs PCR panel and LC-MS analysis, respectively. We found differential expression of miRNAs miR-148b-3p, miR-423-3p, miR29b-3p, miR15b-5p, let-7e-5p, miR126-3p, miR-125b-5p and miR-376c-3p as well as hemoglobin, haptoglobin, protein S and C4-binding protein in healthy controls vs PNH patients. Our results warrant further research and provide new information on the content of exosomes that could play a role in the hypercoagulable state in this disease.

OTHER RELATED OMICS DATASETS IN: PXD011768

ORGANISM(S): Homo sapiens

PROVIDER: GSE122352 | GEO | 2019/03/12

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.

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Real-time quantitative PCR analysis of human exosomes

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