Different expression profiles of TNF-α stimulated IEC-6 cells-derived exosomal miRNAs

Abstract:

Objective: To analyze the differential expression of IEC-6 small intestinal epithelial-derived exosomal miRNAs stimulated by TNF-α. Methods: IEC-6 cells were stimulated with TNF-α (final concentration of 50 ng/mL) for 4h (the TNF-α-exosome group), and the supernatant was collected. The exosomes in the supernatant were extracted by the total exosome isolation kit combined with ultracentrifugation. Transmission electron microscopy, nanoparticle tracking analysis (NTA) and Western blot were performed to identify exosomes. High throughput sequencing was used to compare the differential expression of exosome miRNAs between the control-exosome group (IEC-6 normally incubated with TNF-α-free medium) and the TNF-α-exosome group. Target Scan, miRDB and miRWalk were used to predict target genes of differential miRNAs, cytoscape was applied for the construction of miRNAs-target genes network and DAVID annotation tools were applied on target genes to analyze the biological functions of genes. Results: The transmission microscopy showed that there were a large number of exosomes, which were round or elliptical lipid bilayer vesicles, heterogeneously distributed, with a diameter about 50 nm to 200 nm. NTA showed that the main peak of the particle size was 141.9 nm, the concentration was 3.41×10⁹particle/mL. Western blot results showed that the exosome marker protein CD63 was positive. High-throughput sequencing screened six differentially expressed exosomal miRNAs (|log₂ (fold change) |>2, P<0.05). Compared with the control-exosome group, rno-miR-125a-5p (|log₂ (fold change) |=3.689 0) and rno-miR-351-5p (|log₂ (fold change) |=3.803 1) in the TNF-α-exosome group were significantly down-regulated, and rno-miR-125b-5p (|log₂ (fold change) |=2.933 0), rno-miR-542-3p (|log₂ (fold change) |=2.995 6), rno-miR-30c-5p (|log₂ (fold change) |=3.065 0) and rno-miR-20a-5p (|log₂ (fold change) |=2.890 8) in the TNF-α-exosome group were obvious down-regulated. Six differentially expressed miRNAs target genes were predicted by using the miRNA target gene prediction website and 158 genes were obtained and the network of six miRNAs were constructed. DAVID annotation showed that the target genes of down-regulated miRNAs were mainly involved in 1 Kyoto encyclopedia of genes and genomes (KEGG) pathway and 12 gene ontology_biological process (GO_BP), 15 gene ontology_cell component (GO_CC), 3 gene ontology_molecular function (GO_MF). Conclusion: Exosomes derived from TNF-α-induced small intestinal epithelial cells contain rich miRNAs, and their expression profiles are significantly different. With the help of high throughput sequencing and related database, more information about the exosomal miRNAs derived from small intestinal epithelial cells and its function are revealed, and a new perspective for the pathogenesis of small intestinal mucosal injury-related diseases are provided.