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Effect of knockdown of miR-BART19-3p on gene expression of AGS EBV cells

ABSTRACT: EBV microRNAs, especially rightward transcript (BART) microRNAs play key roles in the tumor growth of EBVaGC. However, the effects of EBV-miR-BART19-3p in EBVaGC remain largely unknown. Here, we report that EBV-miR-BART19-3p is highly expressed in EBVaGC and promoted the proliferation of EBV-associated gastric cancer cells in vitro and accelerate xenograft tumor growth in vivo. Molecularly, EBV-miR-BART19-3p directly targets the growth arrest and DNA damage-inducing protein Beta mRNA (GADD45B) and downregulates its protein expression, which consequently reduce cell cycle G2/M phase arrest. Reconstitution of GADD45B can rescue the proliferation phenotype caused by EBV-miR-BART19-3p in EBVaGC cells, highlighting the role of GADD45B in EBV-miR-BART-driven Gastric cancer. Our study indicated Epstein-Barr Virus MicroRNA BART19-3p played an oncogenic role by a novel mechanism of inhibiting GADD45B in EBVaGC, which provides new insights and might be a potential therapeutic strategy for EBVaGC patients.

ORGANISM(S): Homo sapiens

PROVIDER: GSE302909 | GEO | 2025/07/22

REPOSITORIES: GEO

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Project description:[Aim] EBVaGC accounts for about 10% of gastric cancer and has a better prognosis than EBVnGC, but its molecular mechanism is still unclear. We investigated the role of EBV-miR-BART1-3p in the occurrence and development of EBVaGC by applying bioinformatics methods, high-throughput sequencing, in vitro experiments, target gene screening and verification, differential gene protein interaction network and gene regulatory network construction. [Materials and Methods] The data set GSE51575 was normalized, and the outlier samples were deleted. Finally, gene expression profiles of 11 EBVaGC and 14 EBVnGC tumor tissues and non-tumor control tissues were obtained. Differential gene function analysis was performed, and molecular regulatory network was constructed. The mRNA expression levels of SNU-719 (EBV-positive gastric cancer cell line), AGS, AGS-NC (EBV-negative gastric cancer cell line) and AGS-OE (lentivirus-transfected miR-BART1-3p overexpression cell line) were detected by high-throughput next-generation sequencing technology, and differential gene expression characteristics were analyzed. To investigate the effect of miR-BART1-3p overexpression on mRNA expression level of gastric cancer cells and construct the protein interaction network of differential genes and the regulatory network of miR-BART1-3p gene. DIANA-MR micro-T, miRnada, miRDB, TarBase v.8, and ViRBase online databases were used to predict the target genes of miR-BART1-3p and GO enrichment analysis of target gene function was performed. Real-time fluorescence quantitative PCR was used to detect the expression levels of miR-BART1-3p and target genes in SNU719, AGS-OE and AGS gastric cancer cells. The effects of overexpression of EBV-miR-BART1-3p on proliferation, apoptosis, invasion, and migration of gastric cancer cells were investigated by CCK-8, cell cloning, cell scratch and Transwell. [Results] By analyzing the gene expression profile of the GSE51575 dataset, we found that EBVaGC up-regulated the expression of immune cell membrane protein receptors and chemokine family, and was involved in inducing tumor immunity, suggesting a better prognosis. EBVnGC significantly up-regulated the expression of AURK, BUB1B, CCNA2 and CDC20, suggesting the risk of malignant progression and recurrence. High-throughput next-generation sequencing of SNU719 and AGS to detect differences in gene expression revealed that EBVaGC cells (SNU719) up-regulated the expression of genes that inhibit malignant progression as well as potential tumor immunotherapy targets. These results are consistent with those found in the GSE51575 dataset. Fifteen miR-BART1-3p target genes, including FAM168A, TET3, HBP1, ZBTB5, ATXN7L3, KMT5C, SPARC and BMF, were predicted and screened by online tools. After the overexpression of miR-BART1-3p in AGC(EBVnGC) cells, the proliferation, migration, and invasion ability were significantly inhibited, and the signaling pathway promoting tumor progression was also limited in activation. [Conclusions] The overexpression of miR-BART1-3p may inhibit the proliferation and invasion and migration of gastric cancer cells by inhibiting the activation of STAT3/ pSTAT3 and PI3K/AKT signaling pathways and may become a target for the treatment of EBVaGC, which is worthy of further study.

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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Effect of knockdown of miR-BART19-3p on gene expression of AGS EBV cells

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