Screening And Identification Of Differentially Expressed MiRNAs In Gastric Cancer Cell-derived Exosomes Induced By Helicobacter Pylori

Objective: Exosomes are another new carrier that mediates cell-cell information communication,and play an important role in the process of immune response,inflammation and tumor,etc.This experiment is intended to isolate and identify Helicobacter pylori(H.pylori)-induced human gastric cancer SGC-7901 cell-derived exosomes,further to screen the differentially expressed microRNA(miRNA)using a microarray chips.Then we aimed to predict the potential target genes of differentially expressed miRNAs and analyze the possible signaling pathways involved.Our results will provide new clues for further elucidating the carcinogenic mechanism of H.pylori.Methods: Human gastric cancer SGC-7901 cells were stimulated with live H.pylori bacteria in vitro for 24 h,and the blank treatment group was used as the negative control.Ultracentrifugation and exosome extraction kit were used to extract the exosomes released by the two groups of cells,and transmission electron microscope(TEM),nanoparticle tracking analysis(NTA)and Western-blot(WB)experiments were employed to identify exosomes.Then,exosomes were labeled with the fluorescent dye PKH67 and co-cultured with THP-1-derived macrophages.The internalization of exosomes by macrophages was observed by laserconfocal fluorescent microscopy.Additionally,miRNA microarray chips were performed to detect the differentially expressed miRNAs of exosomes from the two groups of cells.Real-time fluorescence quantitative PCR(qRT-PCR)was used to verify the expression of four differentially expressed miRNAs.Furthermore,target genes of partial differentially expressed miRNAs(including 30 up-regulated miRNAs and20 down-regulated miRNAs)were predicted by bioinformatics software mirdbv6 and targetscan7.1,and Gene Ontology(GO)functional annotation and Kyoto Encyclopedia of Genes and Genome(KEGG)pathway enrichment analysis were carried out to identify the function of these target genes and possible signal pathways involved.Results:1.The results of NTA showed that the diameter of the isolated exosomes were about 137.1nm,and the particles were revealed as round-shaped vesicles with double layer membrane structure and diameters about 50-150 nm observed through transmission electron microscope(TEM);2.WB experiment found that the expression levels of exosomes marker proteins CD9,CD63 and TSG101 were higher than that in SGC-7901 cells,while the endoplasmic reticulum protein Calnexin was absent in exosomes which can be detected in cells;3.After co-culturing with THP-1 derived macrophages for 12 hours,the exosomes could be internalized by macrophages;4.Compared with the control group,there were 130 up-regulated miRNAs and 111 down-regulated miRNAs in the H.pylori-stimulated group;5.GO functional annotation and KEGG Pathway analysis show that the potential target genes of partial differentially expressed miRNAs mainly regulate RNA metabolism,RNA polymerase II transcription,cell metabolism and protein binding,and participate in PI3K-AKT,NF-κB,JAK-STAT,Stem cell pluripotency and other inflammation and tumor-related pathways.Conclusion: H.pylori treatment caused a significant change in the expression level of exosome miRNAs in SGC-7901 cells,bioinformatics analysis demonstrated that the prospective targets of these differentially expressed miRNAs play an important role in the regulation of inflammation and tumor-related signaling pathways.