MiRNA Sequencing And Bioinformatics Analysis Of EA.hy926 Treated By Oxidative Stress Under Simulated Microgravity

ObjectiveVascular endothelial cells can produce more oxygen free radicals?ROS?,have reduced ability to scavenge ROS in microgravity or simulated microgravity,resulting in increased oxidative stress and dysfunction of endothelial cells.The structural and functional integrity of endothelial cells is essential for vascular function regulation and preventing arteriosclerosis,in which microRNAs?miRNAs?play important role.The study explored the differential expression of microRNAs and their significance in EA.hy926 cells?a fusion of HUVEC and lung cancer cell line A549?induced by oxidative stress under simulated microgravity.MethodsEA.hy926 cells were seeded in cell culture flasks at a density of 10⁵/ml.The cells were divided into 4 groups:control group?C?,control oxidative stress group?C+H₂O₂?,simulated microgravity group?Z?and simulated microgravity with oxidative stress group?Z+H₂O₂?.The Z and Z+H₂O₂ cells were filled with the culture medium?DMEM+10%FBS+1%penicillin-streptomycin?,fixed in a cell clinostat and cultured for 72 hours at 37?with 30 rpm.The cells in group C and C+H₂O₂ were filled with culture medium and placed in a 1G gravity environment with 37?for 72 hours.All cells were then replaced with fresh medium?5ml/vial?,cells in C+H₂O₂ and Z+H₂O₂ cells were treated with hydrogen peroxide at a final concentration of 50 mmol/L for 5 hours,and cells were harvested for subsequent experiments.miRNAs from C,C+H₂O₂,Z and Z+H₂O₂ cells were sequenced by second-generation sequencing technology to screen out the differentially expressed miRNAs.The differentially expressed miRNAs were verified by RT-qPCR.Then,bioinformatics analysis of verified differentially expressed miRNAs with qPCR was carried out to illuminate the functional enrichment,significant signaling pathways,protein-protein interactions,Western blot analysis of core proteins in protein-protein interactions.ResultsDifferentially expressed miRNAs were screened by significant levels P<0.05 and differential folds log ₂ FC>2 or<-2.Compared with the control group,there were 102 differentially expressed miRNAs in the C+H₂O₂ group,in which53 genes were up-regulated,49 genes were down-regulated?C+H₂O₂ vs C?,and the Z group had 97 differentially expressed miRNAs,in which 17 miRNAs were up-regulated and 80 miRNSs were down-regulated?Z vs C?.Z+H₂O₂group had 71 differentially expressed miRNA compared with Z group,in which52 of which were up-regulated and 19 miRNAs were down-regulated?Z+H₂O₂vs Z?.There were 66 differentially expressed miRNA in Z+H₂O₂ group compared with C+H₂O₂ group,in which 38 miRNSs were up-regulated and 28 miRNSs were down-regulated?Z+H₂O₂ vs C+H₂O₂?.The RT-qPCR results showed that the expression of hsa-miR-29b-3p and hsa-miR-200c-3p were increased in the Z+H₂O₂ group compared with the C+H₂O₂ group,which was consistent with the miRNA sequencing results.The hsa-miR-29b-3p target genes are ERCC6,NFIA,TET3,BRWD3,FBN1,TET1,ATAD2B,RNF19A,etc.,and hsa-miR-200c-3p target genes are APOO,OSTM1,PAIP2,SULF1,KIAA1432,KDR,BAP1,DGKA,KLF4,etc.GO functional analysis showed that the two miRNAS were enriched in biological processes such as biological adhesion,cellular component such as cell binding and molecular functional such as cell binding.The KEGG pathway showed that hsa-miR-29b-3p was significantly enriched in signaling pathways such as focal adhesion,hsa-miR-200c-3p was significantly enriched in signaling pathways such as microRNAs in cancer.Protein-interacting analysis indicated that the COL family proteins in the hsa-miR-29b-3p target gene were at a critical position in the interaction,and RHOA in the hsa-miR-200c-3p target gene was at a critical position.The core protein RHOA and BCL-2 in Z+H₂O₂ vs C were confirmed by Western blot had significant expression trend.ConclusionsThe miRNAs in endothelial cells treated by oxidative stress were significantly differentially expressed under simulated microgravity including hsa-miR-29b-3p and hsa-miR-200c-3p which have special regulating role in the physiological function of endothelial cells under simulated microgravity through their target genes and signal pathways.