Analysis Of Differentially Expressed MiRNAs In Septic Acute Kidney Injury Target Oxidative Stress

OBJECTIVE To observe the inflammatory factors and oxidative stress changes in septic AKI patients,identify specific miRNAs involved in sepsis-induced AKI and explore their targeting pathways.METHODS There were 35 septic AKI patients,30 sepsis non-AKI patines and 20 health control included in the study.Serum inflammory factors and 8-iso-prostaglandin F2α levels were detected.The expression profiles of miRNAs in 15 serum samples from patients with sepsis-induced AKI(n=6),sepsis-non AKI(n=6)who were intestinal infection or enterobacteria infection confirmed by blood culture,and healthy volunteers(n=3)were investigated.After having passed RNA quantity measurement,the samples were labeled and hybridized on the miRCURY? LNA Array(v.18.0).Following the washing steps the slides were scanned using the Axon Gene Pix 4000 B microarray scanner.Scanned images were then imported into Gene Pix Pro 6.0 software(Axon)for grid alignment and data extraction.Replicated miRNAs were averaged and miRNAs that intensities≥30 in all samples were chosen for calculating normalization factor.Expressed data were normalized using the Median normalization.After normalization,significant differentially expressed miRNAs were identified through Volcano Plot filtering.Hierarchical clustering was performed to show distinguishable miRNA expression profiling among samples.Furthermore,eight miRNAs were randomly selected out of the differentially expressed miRNAs for further testing by q PCR.Then the targets of the differentially expressed miRNAs were predicted by Target Scan,mirbase and Miranda.Finally,the significant functions and involvement in signaling pathways of gene ontology(GO)and KEGG pathways were analyzed.RESULTS The TNF-α,IL-1β,IL-6,IL-2Sr and IL-10 incresed significantly in septic AKI patients,while IL-8 decreased significantly compared with sepsis non-AKI patients(p<0.05).The difference was not statistically significant between sepsis non-AKI and health control(p>0.05).The 8-iso-PGF2α increased in septic AKI patients compared with sepsis non-AKI group(p<0.05).q PCR analysis confirmed that the expressions levels of hsa-miR-142-5p,hsa-miR-22-3p,hsa-miR-191-5p,hsa-miR-23a-3p and hsa-miR-4456 were significantly lower in patients with sepsis compared with the healthy volunteers,while hsa-miR-4270,hsa-miR-4321,hsa-miR-3165 were higher in the sepsis patients.Statistically,miR-4321 and miR-4270 were significantly upregulated in the sepsis-induced AKI compared with sepsis-non AKI,while only miR-4321 significantly overexpressed in the sepsis groups compared with control groups.GO analysis showed that biological processes regulated by the predicted target genes included diverse terms.They were related to kidney development,regulation of nitrogen compound metabolic process,regulation of cellular metabolic process,cellular response to oxidative stress,mitochondrial outer membrane permeabilization,etc.Pathway analysis showed that several significant pathways of the predicted target genes related to oxidative stress.miR-4321 was involved in regulating AKT1,m TOR and NOX5 expression while miR-4270 was involved in regulating PPARGC1 A,AKT3,NOX5,PIK3C3,WNT1 expression.Function and pathway analysis highlighted the possible involvement of miRNA-deregulated m RNAs in oxidative stress and mitochondrial dysfunction.CONCLUSION This study might help to improve understanding of the relationship between serum miRNAs and sepsis-induced AKI,and laid an important foundation for further identification of the potential mechanisms of sepsis-induced AKI and oxidative stress and mitochondrial dysfunction.