Expression Profile Of MicroRNA In Human Urine Stem Cell Exosomes Under Normoxia And Hypoxia

Objective: The expression profiles of microRNAs in human urine stem cell exosomes under normoxia and hypoxia were compared.Methods: Fresh urine of healthy adult males was collected using an optimized urine collection device.Human urine stem cells(hUSCs)were isolated from urine by "primary centrifugation".They were cultured to P3 and divided into two groups: the normoxic group and the hypoxic 48 h group.Their surface markers were identified by flow cytometry and their growth curves were plotted and compared using the CCK-8 method.Human urinary stem cells(hUSCs)in the normoxia and hypoxia groups were induced and differentiated in vitro by osteogenic adipogenesis.They were identified and compared using Von Kossa,alizarin red,alkaline phosphatase,and oil red O staining.hUSCs-Exo was extracted from the hUSCs culture supernatant of the normoxic group and the hypoxia 48-hour group by the modified ultra-high speed centrifugation method.The morphology of hUSCs-Exo was extracted by transmission electron microscopy.The concentration and size of hUSCs-Exo were analyzed by NTA method.The expression of specific proteins CD9,CD63 and HSP70 were detected by Western blotting.The identified hUSCs-Exo extracted in the normoxic group and the hypoxia group were obtained.This study used the Small RNA Library Prep Kit for Illumina to construct a library,which was then initially quantified using Qubit 3.0.Library size ranges were tested using an Agilent 2100 Bioanalyzer.After the size of the inserted fragment was as expected,the Q-PCR method was used to accurately quantify the effective concentration of the library(library effective concentration 2 nM)to ensure library quality.After the library was qualified,the different libraries were combined according to the effective concentration and the target data amount,and then sequenced using the HiSeq PE150 model.Finally,the miRNA gene sequencing and analysis were performed.Results: The cell morphology was typical "spindle shape" or "fusiform";the cells in hypoxia group grew faster than the cells in normoxia group,and the growth curves of both were "S" shape;the positive expression of hUSCs The surface markers specific to mesenchymal stem cells were CD44,CD73,CD105 and CD90;both normoxia and hypoxia groups had the potential to induce osteogenic and adipogenic differentiation,and the bone effect of Hp-hUSCs was better than that of N-hUSCs.After ultracentrifugation,a white precipitate attached to the hypoxic and hypoxic groups of the bottom side of the centrifuge tube was extracted.Observation under transmission electron microscopy: The shape is mainly a tea-like structure with a uniform shape and a circular shape,and the sample size is between 30 nm and 200 nm.By NTA detection,the vesicle size of the N-hUSCs/Hp-hUSCs group was mainly 30-170 nm,and the concentrations were4.28×107/ml and 2.37×107/ml,respectively,and the hypoxia group was higher than the normoxia group.The results of Western blotting showed that the exosome in the normoxia and hypoxia groups positively expressed the internationally accepted conserved proteins CD9,CD63 and HSP70.A total of 851,664 clean sequences were detected in Hp-hUSCs exosomes by high-throughput sequencing,the number of miRNAs was 1693,and there were 7545238 clean sequences in N-hUSCs exosomes.The number of miRNAs was 1063,among which differentially expressed miRNAs A total of 92 miRNAs with up-regulated expression were found in 37 of the miRNAs that were specifically expressed in 37 hypoxia groups: hsa-miR-132-5p,hsa-miR-4473,hsa-miR-135a-5p,hsa-miR-629-3p and so on.There are 37 miRNAs that are down-regulated.The up-regulated miRNAs with significant differences in expression were hsa-miR-132-5p,hsa-miR-4473,hsa-miR-135a-5p,hsa-miR-129-5p,hsa-miR-185-3p,hsa-miR-375,hsa-miR-135 a,hsa-miR-376a-3p,hsa-miR-493-3p,etc.,wherein hsa-miR-375,hsa-miR-135 a,hsa-miR-376a-are involved in tissue repair.3p,hsa-miR-493-3p,etc.,informatics analysis results suggest that differentially expressed miRNAs are involved in biological processes such as tissue repair,cell proliferation and differentiation,growth cycle,and neuromodulation processes.Conclusion: There are significant differences in the expression profiles of miRNAs inhuman urinary stem cell exosomes under normoxia and hypoxia;...