Mechanism Study Of Mesenchymal Stem Cell-derived Exosomes Alleviating Cisplatin Induced HK-2 Cell Damage By Loading MiR-1184

Research background:Acute kidney injury（AKI）is a wide range of clinical syndromes characterized by a sudden decrease in glomerular filtration rate followed by an increase in blood creatinine and urea nitrogen,accompanied by oliguria.Cisplatin is a commonly used chemotherapy drug.One of the side effects is AKI.In previous laboratory studies,we found that Mesenchymal Stem Cells（MSC）-derived exosomes alleviated renal fibrosis by loading miRNA Let-7i-5p antagomir and miR-374a-5p.Adipose Stem Cells（ADSCs）-derived exosomes can improve diabetic nephropathy（DN）by promoting autophagy flow in podocytes and inhibiting apoptosis.ADSCs-derived exosomes carrying miR-215-5p can alleviate the Epithelial-Mesenchymal Transition（EMT）of DN podocytes.It can be seen that stem cell derived exosomes have an excellent effect in the treatment of kidney injury.miR-1184 plays an important role in cell proliferation,cell cycle,inflammation and apoptosis.However,whether MSC-derived exosomes loaded with miR-1184 have a protective effect on AKI remains unknown.Research objectives:Inflammation,increased apoptosis and cell cycle arrest are important pathological processes in the progression of AKI.More and more studies have shown that stem cell-derived exosomes can alleviate kidney injury and delay kidney disease progression by loading miRNAs.Domestic and foreign researches have shown that miR-1184 can inhibit the proliferation,migration and invasion of cancer cells.And in patients with myocardial infarction,miR-1184 can inhibit the apoptosis of myocardial cell.At the same time,our study found that miR-1184 was significantly down-regulated in patients with AKI,which may be an effective target for the treatment of AKI.Therefore,we aimed to investigate whether MSC-derived exosomes loaded with miR-1184 can reduce cisplatin-induced AKI and the related mechanism.Research methods:In our preliminary works,the differentially expressed miRNAs in patients with AKI were first explored by bioinformatics analysis,and their targets were predicted by database.Exosomes were extracted from the supernatant of MSC culture medium by overspeed centrifugal method and identified by Nanoparticle Tracking Analysis（NTA）and immunofluorescence assay.In order to simulate AKI in vitro,cisplatin was used to induce human proximal tubular cell line（HK-2）damage.Cell viability,apoptosis and cell cycle were detected by Cell Counting Kit 8（CCK8）and flow cytometry.Reverse transcription quantitative PCR（RT-q PCR）was used to detect gene expression levels in cells.Western Blot was used to detect protein expression levels in cells and exosomes.Enzyme Linked Immunosorbent Assay（ELISA）was used to detect the expression of inflammatory cytokines in the supernatant of cell culture.All data were represented by mean±SD and analyzed using Graph Pad Prism 7 software.Unpaired Student’s t test was used to analyze the differences between the two groups.Univariate ANOVA analysis and Tukey’s test were used to compare the results of multiple groups,and P<0.05 was considered statistically significant.Research results:1.The results of bioinformatics analysis showed that the expression of miR-1184 was significantly down-regulated in patients with AKI,suggesting that miR-1184 may play an important physiological role in the body.2.Exosomes isolated from MSC with miR-1184 agomir transfection(MSC-Exo^{miR-1184agomir})significantly improved the cisplatin induced decline in cell viability by inhibiting cell apoptosis and cell cycle arrest.The inflammatory response of HK-2 cells induced by cisplatin was significantly inhibited by mediating TNF-αand IL-1β.3.FOXO4 is the target of miR-1184.Overexpression of FOXO4 significantly reversed the effect of MSC-Exo^{miR-1184 agomir} on the levels of inflammatory cytokines secreted by cisplatin-induced HK-2 cells.Conclusions:In a word,our study demonstrated that exosomes derived from MSC significantly ameliorated cisplatin-induced injury in HK-2 cells by delivering miR-1184from the aspects of apoptosis,cell cycle and inflammation,which may provide a new direction for the treatment strategy of AKI in the future.