Molecular Profiles Of MiRNAs In Exosomes Derived From Human Umbilical Cord Mesenchymal Stem Cells To Hypoxia

Objective:To analyze the effect of hypoxia on the molecular profiles of microRNAs(miRNAs)in exosomes derived from human umbilical cord mesenchymal stem cells(UMSCs).and to explore the potential mechanisms and therapeutic targets of hypoxia-preconditioned UMSCs for myocardial infarction repair.Methods:Firstly,the hypoxia model of UMSCs was established,and the hypoxia effect was verified by detecting the expression levels of hypoxia-related genes by qPCR.Then,the hypoxia group and normoxia group were compared in terms of the morphology,particle size and concentration of exosomes.Then,the miRNAs in the exosomes of the hypoxia and normoxia groups were sequenced and analyzed to compare the differences in the miRNA molecular profiles between the two groups.Finally,according to the sequencing results,functional experiments were conducted to verify.Results:By comparison,we found that hypoxia state had no significant effect on the morphology,particle size and concentration of UMSCs-derived exosomes.The sequencing results showed that the bias of the first base of miRNAs of certain lengths in exosomes changed significantly to hypoxia.There were significant differences in the expression of miRNAs to hypoxia,and a total of 25 differential miRNAs were screened.GO functional enrichment analysis revealed that these differential miRNAs were mainly related to metabolic processes.KEGG pathway analysis found that the differential miRNAs were most significantly enriched in lysosome,cell autophagy and other related pathways.CCK-8 cell viability assay showed that the cell viability of 5 μM and 25 μM groups increased significantly after 24 and 48 h treatment with Rapamycin,the autophagy activator.The cell viability of UMSCs treated with autophagy inhibitor 3MA for 24 h and 48 h showed no significant difference compared with the control group.The activity of Caspase3/7,a protein associated with apoptosis,was determined.After 24 h and 48 h treatment with Rapamycin,the activity of Caspase3/7 in 5μM and 25 μM groups was significantly decreased compared with the control group.However,there was no significant difference in Caspase3/7 activity between the treatment group and the control group after 24 h and 48 h treatment with the autophagy inhibitor 3-MA.Conclusion:Hypoxia had no significant effect on the morphology,particle size and concentration of UMSCs-derived exosomes.Hypoxia significantly changed the first base bias of miRNAs in UMSCs-derived exosomes.which in turn changed the expression patterns of miRNAs.These differential miRNAs are functionally involved in cellular metabolic processes,and are related to signaling pathways such as lysosomerelated functions and the regulation of autophagy.Activation of autophagy can enhance the cell viability and inhibit the apoptosis of UMSCs.Inhibition of autophagy had no significant effect on the viability and apoptosis of UMSCs.