The Role Of MiR-107-mediated Regulation Of RPS19 In The Treatment Of AKI By Mesenchymal Stem Cells Based On Bioinformatics Methods

Part one:MicroRNAs identification,target gene prediction and DEGs screeningPurpose:The role of mesenchymal stem cells(MSCs)in the repair of acute kidney injury has been extensively studied.Yet,some underlying molecular mechanisms remain unclear.In this part of the study,published microRNA(miRNA)transcripts were combined with quantitative proteomic data to reveal any distinct miRNA or gene in the treatment of AKI by MSCs.Methods:1.Microarray data were downloaded from the study of Yuan Zhu et al..Data consisted of miRNAs in renal tissues from AKI mice that did or did not undergo MSCs treatment.Kidney tissues showed differential expression among 44 miRNAs(36 increased,8 decreased).2.Proteomics resources from the following cells were downloaded:a human renal proximal small tube cell line(HK-2)that was subjected to chemical anoxia followed by a 3-h recovery period compared with that of renal cells subjected to anoxia and co-cultured with hMSC during the recovery period.Among them,262 proteins showed higher expression,while,48 proteins showed lower expression.3.Using five verified miRNA target prediction programs(miRanda-mirSVR,miRDB,miRWalk,RNA22v2 and TargetScan6.2),bioinformatics predictions were performed on 44 miRNAs-regulated target genes.4.The target genes predicted by miRNA and the differentially expressed proteins in HK-2 cells were analyzed in depth.The Venn diagram of target genes and differentially expressed proteins was drawn by bioinformatics analysis method.The most commonly differentially expressed genes(DEGs)after intervention of MSCs in the two groups were obtained.Results:1.We inputted the 44 miRNAs into the miRanda-mirSVR,miRDB,miRWalk,RNA22v2 and TargetScan6.2 databases to predict the regulated genes;from which,15270 target genes were identified.2.Following a careful analysis of the 15270 predicted target genes and the 310 differentially expressed proteins,we ultimately screened 155 target DEGs after MSCs intervention in AKI model in vitro and in vivo.Summary:The transcriptomic data obtained from Cisplatin induced AKI rats treated by MSCs were intersected with the proteomics datas obtained from HK-2 cells,and finally 155 target DEGs were obtained.Part two:The GO enrichment,KEGG pathway analyses and PPI network construction of target differentially expressed genesPurpose:In the first part,the common differentially expressed genes after MSCs treatment in AKI were filtered.Through bioinformatics analysis of related genes and proteins,more comprehensive gene-protein interaction network and annotation information can be obtained to further explore the key gene regulated by miRNA.Methods:1.The GO and KEGG pathway analyses were derived from the Database for Annotation,Visualization and Integrated Discovery(DAVID).In this experiment,DEGs were uploaded into DAVID to carry out correlation analyses of relevant GO and KEGG pathways.The cutoff criterion was set as P<0.05.2.All candidate DEGs were entered into the STRING website,and a confidence score>0.9 or a cutoff criteria of 0.98 was set for the protein-protein interaction(PPI)network foundation.Results:1.GO enrichment analysis revealed that a significant number of the DEGs were condensed in extracellular vesicles,extracellular organelles,extracellular exosomes,vesicles,membrane-bound vesicles,extracellular regions,cytosol,RNA binding,poly(A)RNA binding and extracellular regions.2.According to KEGG pathway analysis,the main enriched signal pathways of DEGs were obtained.It was found that ribosome pathway may play an important role in MSCs treatment of AKI.3.Using a confidence score>0.9,the PPI network of DEGs was established and consisted of 105 nodes and 357 edges.A total of 27 central node genes were screened out by screening>10 degrees in 105 nodes.4.Another PPI network with 20 nodes and 51 edges was constructed using the cutoff combinatorial score>0.98.Of the 20 nodes,10 central node genes were screened according to the>9 degree criteria,which all encode ribosomal proteins.Summary:The 155 target differential genes were analyzed by GO enrichment analysis,KEGG pathway analysis and PPI network construction.The results showed that ribosome pathway may play an important role in MSCs treatment of AKI,and RPS19 may be the key ribosome protein in AKI.Part three:Mesenchymal stem cells alleviate acute kidney injury via miR-107-mediated regulation of ribosomal protein S19Purpose:Identification of ribosomal protein S19 as direct target of miR-107 was further verified.Expressions of miR-107,RPS19,PCNA and Bax/Bcl-2 were respectively detected in cisplatin induced AKI rats and HK-2 cells,in order to explore the possible mechanism of MSCs in the treatment of acute kidney injury.Methods:1.The RPS19 3’-UTR,including the miR-107 targeting sequence,was cloned into a dual-luciferase reporter plasmid.HEK293 cells were cultured and then transfected for 48h with a double luciferase reporter plasmid and either miR-107 analog or miR-107 inhibitor.The luciferase activity in the transfected cells was determined by a Dual-Luciferase Reporter Assay System.2.We induced a cisplatin-induced AKI rat model,which was divided into normal control group,cisplatin group and cisplatin+MSCs group.The levels of serum creatinine(Scr)and BUN were tested in each group,and pathological changes were observed by PAS staining of renal tissue.Moreover,we tested miR-107 levels using quantitative RT-PCR and measured the expression levels of RPS19 by Western blot(WB).3.We used cisplatin to induce acute injury of HK-2 cells,which were divided into the normal control group,cisplatin group and MSCs group.Morphological changes of cells in each group were observed,and the expression levels of miR-107,RPS19,Bax,Bcl-2 and PCNA were detected by real-time quantitative PCR and WB,respectively.4.HK-2 cells transfected with scramble fragment or miR-107 antisense oligonucleotide using Lipofectamine RNAiMAX were divided into negative control(NC)group and inhibitor group.After treatment with cisplatin,the expression levels of miR-107,RPS19,Bax,Bcl-2 and PCNA in the two groups were evaluated.Results:1.We can see that inhibiting miR-107 expression in HEK293 cells improved the luciferase activity from the plasmid containing the RPS19 3’-UTR.In addition,there was no significant difference in luciferase activity among the mutant groups.These findings suggested that miR-107 can interact with RPS19 mRNA in detail and inhibit the expression of RPS19 at the posttranscriptional level.2.The rats’ kidenys in the cisplatin group showed obvious pathological changes and the levels of serum creatinine(Scr)and BUN in AKI rats were significantly higher than those in the control group.MSCs transplantation significantly alleviated renal tubular injury and decreased Scr and BUN levels in AKI rats.3.Using quantitative RT-PCR and WB,we confirmed the increase in miR-107 levels and the decrease in the expression of RPS19 protein in the kidneys of AKI rats.Bone marrow mesenchymal stem cells could mitigate these changes.4.miR-107 expression in HK-2 cells increased after cisplatin was added and was down-regulated when the cells were co-cultured with MSCs.We also found that the expression levels of RPS19 and PCNA in the cisplatin group were lower than those in the control group,and the ratio of apoptosis-related protein Bax/Bcl-2 was increased.The expression levels of RPS19 and PCNA in MSCs group were significantly increased,and the Bax/Bcl-2 ratio was down-regulated.5.We also detected the expression levels of RPS19 in HK-2 cells treated by cisplatin from NC group and inhibitor group.After inhibiting miR-107 expression,the expression of RPS19 was increased,and at the same time,PCNA expression was higher and Bax/Bcl-2 ratio was decreased.Summary:1.The dual-luciferase reporter gene assay confirmed that RPS19 was a direct regulatory target of miR-107 during AKI.2.Studies on cisplatin-induced AKI rats and HK-2 cells showed that miR-107 level in cisplatin group was significantly higher than that in the control group,and the expression level of RPS19 protein was down-regulated.However,MSCs can reverse these changes.In addition,we further verified that MSCs could up-regulate PCNA expression and down-regulate Bax/Bcl-2 ratio in HK-2 cells.3.MiR-107 antisense oligonucleotide was used to prove the negative regulatory effect of miR-107 on RPS19 in HK-2 cells.The findings show that miR-107 may target RPS19 in response to potential apoptosis in cisplatin-injured HK-2 cells.