| ObjectiveTo study the effects and potential mechanism of lipopolysaccharide(LPS)-induced exosomes derived on proliferation and migration of IEC-6 cells via miR-218a-5p,and also illuminate the effect of glycyrrhizic acid via this pathway.Methods1.IEC-6 cells-derived exosomes were isolated by the kit combined with ultracentrifugat ion,which were identified by TEM,NTA,and West ern blot.2.High-throughput sequencing was used to analyze differentially expressed profiles of miRNAs in induced IEC-6 cells-derived exosome,and G0 and KEGG enrichment analysis of di fferentially expressed miRNAs in samples was performed to predict possible signal pathways.3.To analyze exosome-cell affinity,the intake of exosomes was measured by flow cytometry after IEC-6 cells-derived exosomes labeled with Exo-Green fluorescence and incubated with cells.The effects of LPS-stressed exosomes and its different ial miR-218a-5p on the proliferation and migration capacity of IEC-6 cells were measured by EdU and MTT assay and wound healing assay respectively.Exo-Fect TM exosomal transfection ki t,was used to transfect miR-218a-5p into exosomes.The transfected exosomes were co-incubated with IEC-6 cells,and wound healing assay was used to detect effect of miR-218a-5p-transfected exosomes in IEC-6 cells migrat ion.4.The potential target genes of rno-miR-218a-5p were predicted and analyzed by three bioinformatics softwares.FERMT3 and CaMK Ⅱ γ mRNA expression were detected by RT-qPCR and the expression of FERMT3,CaMKⅡ γand Integrin α4 protein were detected by Western blot.5.The effect of gl ycyrrhizic acid-acted exosomes on the proliferation and migration capacity of IEC-6 cells were measured by EdU and wound healing assay.All-in-One TM miRNA qRT-PCR was used to quantify expression of rno-miR-218a-5p in glycyrrhizic acid-acted exosomes.Results1.TEM results showed exosomes that uniformly sized,round or circular bilayer lipid membrane structure,saucer-like microvesicles,and with a diameter of about 40 to 120 nm.NTA results showed that the main peak of exosomal particles size was at 155 nm,and the overall concentration was 2.82×109 particles/mL.Western blot results showed positive expression of surface marker protein CD63 in exosomes.These results suggested that the exosome isolation method by using exosome isolation kit combined with ultracentri fugation.2.High-throughput sequencing analysis results showed that 198 differentially expressed miRNAs(|log2(Fold Change)|>1)were det ected in the exosomes of LPS-induced and Control group.Compared with the Control-exosome group,LPS-exosome group has 19 miRNAs were up-regulated,and 179 miRNAs were down-regulated.9 miRNAs were up-regulated and 12 miRNAs were down-regulated among 21 remark differentially expressed miRNAs(the difference was statistically significant(P<0.05)and(|log2(Fold Change)|>1).Compared with the Control-exosome group,the expression of rno-miR-218a-5p in the LPS-exosome group was the highest differential miRNAs,which was 2.5 times to the control,and the expression was significantly up-regulated(P<0.01).The signaling pathways predicted by G0 and KEGG enrichment analysis mainly include Wnt signaling,axon guidance,neurotrophic factor signaling,focal adhesion,leukocyte transmembrane migration,chemokines and vascular endothelial growth factors,etc.It is suggested that differential miRNAs in LPS induced IEC-6 cells derived-exosome maybe involved in cell migration.3.Uptake rate of fluorescent-labeled exosomes by IEC-6 cells was as high as 99.1%,indicating that IEC-6 cel ls-derived exosomes had the strongest affinity for their derived cells.There was no change in proliferation of IEC-6 cells between the Control group and LPS-stressed group after the IEC-6 cells were treated separately for 24 and 48 hours.The result was showed in proliferation of IEC-6 cells after miR-218a-5p mimic and inhibitor treatment.Wound healing assay results showed that compared with the Control-exosome group,LPS-stressed exosomes promote IEC-6 cell migration with significant differences(P<0.05).Compared with the mimic negative control(NC)group,miR-218a-5p mimic enhances IEC-6 cell wound healing capaci ty and promotes cell migration(P<0.05).Compared with respective control transfected exosome,t he miR-218a-5p mimics transfected exosome promoted IEC-6 cell migration(P<0.05).Meanwhile,the effect of migration-promoting of LPS-stressed exosome was antagonized after transfected into miR-218a-5p inhibitor(P<0.05).It is suggested that LPS-stressed exosomes may promote IEC-6 cell migration by mediating miR-218a-5p.4.Bioinformatics software prediction results showed that FERMT3 may be a potential target gene of miR-218a-5p,and related to cel l migration.FERMT3 mRNA and protein were significantly down-regulated(P<0.05)in IEC-6 cells after transfection with miR-218a-5p mimic.Integrin α4 protein expression was significantly down-regulated(P<0.05),as well as CaMK Ⅱ γ mRNA and protein expression were significantly down-regulated(P<0.01).Wound healing assay results showed that si lencing CaMK Ⅱ γ protein could promote IEC-6 cell migration(P<0.05),compared with the siRNA negative control(siR-NC)group,suggesting that miR-218a-5p may promote IEC-6 cell migration by silencing CaMK-Ⅱ γ protein.5.EdU assay showed that compared with the Control-exosome group,the cell proliferation rate of the glycyrrhizic acid-acted exosomes group did not changed,indicating that glycyrrhizic acid-acted exosomes had little effect on the proliferation of IEC-6 cells.Wound healing assay showed that compared with the Control-exosome group,glycyrrhizic acid-acted exosomes promoted IEC-6 cell migration(P<0.05).(Glycyrrhizic up-regulated the expression of rno-miR-218a-5p in IEC-6 derived exosomes(P<0.05).Conclusion1.IEC-6 cells-derived exosomes were isolated successfully with good purity by the kit combined with ul tracentrifugation.2.High-throughput sequencing and G0 and KEGG analysis showed that the expression of rno-miR-218a-5p was high and signi ficantly up-regulated in LPS induced IEC-6 cells-derived exosomes,and rno-miR-218a-5p may be involved in cell migration.3.LPS-stressed exosomes and their differential miR-218a-5p can promote IEC-6 cell migration,and miR-218a-5p was involved in the promotion of migration by LPS-stressed exosomes in IEC-6 cells.4.miR-218a-5p may promote IEC-6 cell migration by down-regulating the expression of FERMT3,Integrin α4 and CaMKⅡ γ.5.Glycyrrhizic acid-acted exosomes can promote IEC-6 cell migration,which may be related to its up-regulation of miR-218a-5p. |
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