Microarray analysis of long noncoding RNAs in exosomes from SNU-668 Erastin-resistant cells and parental SNU-668 cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE212303 | GEO | 2022/09/02
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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Project description:As a type of secreted membrane vesicle, exosomes are emerging as an important mode of cell-to-cell communication. The objective of this study was to compare the abundance of transcripts present in the parental B16F0 cell to transcripts present in exosomes isolated from B16F0 conditioned media. Identifying local mechanisms of immunosuppression is a key barrier for expanding the clinical benefit of cancer immunotherapy. While exosomes are emerging as a new mode of intercellular communication, their role in establishing a malignant tissue niche remains unclear. Similar to the sculpting of tumor antigens during oncogenesis, a related hypothesis is that proteins secreted by malignant cells are shaped by somatic evolution. To test this hypothesis, we characterized the biological influence of tumor-derived exosomes on immune cell function. In particular, we analyzed exosomes from three melanoma models: B16F0, a non-immunogenic model of malignant melanoma; Cloudman S91, a model of immunogenic melanoma; and Melan-A, an immortalized melanocyte cell line. Using electron microscopy, exosomes derived from all three cell lines were morphologically similar. The exosomes contained receptors derived from the parent cell as demonstrated by IL12RB2 expression on B16F0 exosomes and intact mRNAs. Furthermore, transcript profiling of B16F0 exosomes and cells suggested that exosomal mRNA is enriched for mRNAs that target immune-related pathways, including Ptpn11 that inhibited T cell proliferation and Dnmt3a that inhibited T cell production of IFN-gamma. Functionally, B16F0 exosomes dose-dependently suppressed cell proliferation and the expression of IL12RB2 in primary CD8+ T cells. In contrast, Cloudman S91 exosomes promoted T cell proliferation and Melan-A exosomes had a negligible effect on primary CD8+ T cells. Collectively, the results are consistent with somatic editing of exosomal payloads and suggest that exosomes establish a density-dependent field effect by altering the activity of immune cells that enter the tumor microenvironment.
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