Project description:Genome-wide mRNA expression profiling was performed in AGS, gastric cancer cell line, upon miR-25 silencing. At 48 hours upon anti-miR-25-3p (miRNA inhibitor) and non-targeting control RNA transfection, the whole transcriptome profiling was performed in triplicates. The miR-25 silencing elevates the diffuse gastric cancer features like expression of COL1A2, expression of COL1A2 co-expressed genes, Epithelial to Mesenchymal Transition (EMT) and angiogenesis associated genes.
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:Gastric cancer is one of the most common cancers worldwide. Epstein-Barr virus-associated gastric cancer accounts for approximately 10% of all gastric cancers. EBV expresses its own proteins and miRNAs (BART miRNAs) and regulates host gene expression. In this study, we examined the effect of EBV infection on host mRNA expression. Differential gene expression was analyzed between EBV-negative human gastric cancer cell line AGS and EBV-positive human gastric cancer cell line AGS-EBV.
Project description:Persistent colonization of the gastric mucosa by Helicobacter pylori (Hp) elicits chronic inflammation and aberrant epithelial cell proliferation, which increases the risk of gastric cancer. We examined the ability of microRNAs to modulate gastric cell proliferation in response to persistent Hp infection and found that epigenetic silencing of miR-210 plays a key role in gastric disease progression. Importantly, DNA methylation of the miR-210 gene was increased in Hp-positive human gastric biopsies as compared to Hp-negative controls. Moreover silencing of miR-210 in gastric epithelial cells promoted proliferation. We identified STMN1 and DIMT1 as miR-210 target genes and demonstrated that inhibition of miR-210 expression augmented cell proliferation by activating STMN1 and DIMT1. Together, our results highlight inflammation-induced epigenetic silencing of miR-210 as a mechanism of induction of chronic gastric diseases, including cancer, during Hp infection. To identify miR-210 targets in gastric cells, whole transcriptome analysis of AGS and MKN45 cells transfected with pre-miR-210 was conducted using Affymetrix GeneChip Human Genome U133 Plus 2.0 Array.
Project description:Ornithogalum is one of the therapeutic formulation used in homeopathic treatments. It is specifically used in the treatment for gastric and duodenal ulcerations. Towards understanding the anticancer mechanism, we investigated the genome-wide mRNA changes upon treating AGS Gastric Cancer cells with Ornithogalum. We observed that totally 707 genes were significantly regulated upon Ornithogalum Treatment, among them 246 genes were upregulated and 461 genes were downregulated. The results provide insight into molecular implication and gene level expression of AGS upon treatment with Ornithogalum. Total RNA was isolated from AGS gastric cancer cells treated with 0.01% of ornithogalum and ethanol control and profiled using Affymetrix Human Gene 1.0 ST Array (HuGene-1_0-st).
Project description:We investigate the expression of miRNA in exosome of EBV-positive gastric carcinoma cells. The exosomes of EBV-positive and negative gastric carcinoma cells were separated by ultracentrifugation, the morphology of exosomes was identified by transmission electron microscopy, the exosome size was analyzed by Nanosight, and the expression of exosome membrane protein CD63 and CD81 was detected by western blot. High-throughput sequencing was used to detect miRNA expression profiles in gastric cancer cell lines and their exosomes. Under the ultra-microscopic electron microscope, the exosomes are seen as a typical translucent cup-like structure or a flat spherical structure. The nanoparticle tracking analyzer (Nanosight) showed that the exosomes were between 30 and 150 nm in diameter. Western blot(WB) assays showed that exosomes secreted by EBVaGC and EBVnGC cells expressed specific exosome membrane-associated proteins CD63 and CD81. High-throughput sequencing revealed that EBVaGC(SNU-719) and EBVnGC(AGS) and their secreted exosomes were highly expressed with certain human miRNAs, among which AGS-exo was highly expressed with hsa-miR-23b-3p, hsa-miR-320a-3p, and hsa-miR-4521. SNU-719-exo was highly expressed as hsa-miR-21-5p, hsa-miR-148a-3p and hsa-miR-7-5p. Nearly all EBV-related miRNAs (EBV-miRNA) were expressed in SNU-719 cells and their exosomes, among which EBV-miR-BART1-5p, EBV-miR-BART17-3p and EBV-miR-BART18-5p were the highest in SNU-719 cells, EBV-miR-BART1-5p, EBV-miR-BART18-5p and EBV-miR-BART17-3p were the highest in SNU-719-exo.
Project description:Revealing Dominant Regulatory MicroRNA-495-3p that Governs Multiple Epigenetic Modifiers in Gastric Carcinogenesis In this study, we identified miR-495-3p targeting multiple epigenetic modifiers through comprehensive miRNA and mRNA profiling analysis with in silico target prediction in GC. Western blotting assay or quantitative real time PCR was performed to confirm the expression of miR-495-3p and targets of it. We applied miRNA mimics to ectopic overexpression in gastric cancer cells and observed tumor suppressive effects of miR-495-3p in the growth and metastasis of cancer. Also, we confirmed the status of CpG islands of miR-495-3p promoter using methylation specific PCR analysis.
Project description:Ornithogalum is one of the therapeutic formulation used in homeopathic treatments. It is specifically used in the treatment for gastric and duodenal ulcerations. Towards understanding the anticancer mechanism, we investigated the genome-wide mRNA changes upon treating AGS Gastric Cancer cells with Ornithogalum. We observed that totally 707 genes were significantly regulated upon Ornithogalum Treatment, among them 246 genes were upregulated and 461 genes were downregulated. The results provide insight into molecular implication and gene level expression of AGS upon treatment with Ornithogalum.