Lin Mengyao, Hu Shun, Zhang Tianyi, Li Jiezhen, Gao Feng, Zhang Zhenzhen, Zheng Ke, Li Guoping, Ren Caihong, Chen Xiangna, Guo Fang, Zhang Sheng
Department of Pathology, The First Affiliated Hospital, Fujian Medical University, 20 Chazhong Road, Fuzhou 350005, China.
Department of Pathology, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, 999 Huashan Road, Fuzhou 350212, China.
Cancers (Basel). 2023 May 19;15(10):2841. doi: 10.3390/cancers15102841.
EBV encodes 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.
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.
The exosomes secreted by EBV-positive and negative gastric cancer cells range in diameter from 30 nm to 150 nm and express the exosomal signature proteins CD9 and CD63. Small RNA sequencing showed that exosomes expressed some human miRNAs, among which hsa-miR-23b-3p, hsa-miR-320a-3p, and hsa-miR-4521 were highly expressed in AGS-exo; hsa-miR-21-5p, hsa-miR-148a-3p, and hsa-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 FAM168A, MACC1, CPEB3, ANKRD28, and USP37 after screening by a 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 were 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.
miR-BART1-3p can affect the growth of tumor cells through the 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.
EBV编码至少44种参与免疫调节和疾病进展的miRNA。外泌体可作为EBV-miRNA-BART细胞间传递的载体，并影响细胞的生物学行为。我们对外泌体进行了表征，并建立了外泌体共培养实验，以探讨miR-BART1-3p通过外泌体途径传递的机制及其对肿瘤细胞增殖和侵袭的影响。
通过透射电子显微镜、纳米可视技术和蛋白质印迹法对EBV阳性和EBV阴性胃癌细胞的外泌体进行表征，并用高通量测序外泌体中的miRNA表达谱。将miR-BART1-3p高表达或低表达的外泌体与AGS细胞共培养，研究其对胃癌细胞增殖、侵袭和迁移的影响。通过PITA、miRanda、RNAhybrid、virBase和DIANA-TarBase v.8数据库筛选和预测EBV-miR-BART1-3p的靶基因，并通过qPCR检测共培养后靶基因的表达。
EBV阳性和阴性胃癌细胞分泌的外泌体直径在30nm至150nm之间，并表达外泌体标志性蛋白CD9和CD63。小RNA测序显示外泌体表达一些人类miRNA，其中hsa-miR-23b-3p、hsa-miR-320a-3p和hsa-miR-4521在AGS-exo中高表达；hsa-miR-21-5p、hsa-miR-148a-3p和hsa-miR-7-5p在SNU-719-exo中高表达。所有EBV miRNA均在SNU-719细胞及其外泌体中表达，其中EBV-miR-BART1-5p、EBV-miR-BART22和EBV-miR-BART16在SNU-719细胞中表达最高；EBV-miR-BART1-5p、EBV-miR-BART10-3p和EBV-miR-BART16在SNU-719-exo中表达最高。胃癌细胞中miR-BART1-3p沉默后，肿瘤细胞的增殖、愈合、迁移和侵袭能力显著提高。激光共聚焦显微镜显示外泌体可将miRNA携带到受体细胞中。与miR-BART1-3p沉默的外泌体共培养后，胃癌细胞的增殖、愈合、迁移和侵袭能力显著提高。通过靶向数据库筛选，miR-BART1-3p的靶基因是FAM168A、MACC1、CPEB3、ANKRD28和USP37。CPEB3在所有外泌体共培养细胞中均不表达，而ANKRD28、USP37、MACC1和FAM168A均有不同程度的表达。miR-BART1-3p上调后，USP37和MACC1表达下调，这可能是miR-BART1-3p通过外泌体调节胃癌细胞增殖的关键靶基因。
miR-BART1-3p可通过外泌体途径影响肿瘤细胞的生长。与miR-BART1-3p沉默表达的外泌体共培养后，胃癌细胞的增殖、愈合、迁移和侵袭能力显著提高。USP37和MACC1可能是miR-BART1-3p调节细胞增殖的潜在靶基因。
