The Role Of Exosomes Derived From Induced Pluripotent Stem Cells In Cardiomyocyte Injury In Vitro And MiRNA Profiling

[Objective:]Acute myocardial infarction(AMI)is myocardial necrosis caused by acute persistent coronary ischemia and hypoxia,which ultimately leads to myocardial remodeling and dysfunctional cardiac function.At present,the treatment of myocardial infarction mainly includes drugs,thrombolysis,intervention and surgery,but these measures can not fundamentally solve the problem of myocardial repair,so regeneration and repair therapy plays a crucial role in myocardial infarction.At present,stem cell therapy is regarded as a promising method to treat myocardial infarction.At the same time,based on the paracrine function of stem cells,the repair effect of stem cell derived exosomes has become a research hotspot in acute myocardial injury.Induced pluripotent stem cell(iPSC)is a kind of totipotency stem cells which is similar to embryonic stem cells by reprogramming factors into adult cells.Based on its wide sources,iPSC can eliminate the problems of immune rejection and ethics.It has a wide application prospect in the treatment of repairing damaged tissues and organs,but it still faces the effect of tumorigenicity in cell therapy.In the study of exosomes,based on the similarity between induced pluripotent stem cells and embryonic stem cells,it has been found that induced pluripotent stem cells derived exosome(iPSC-Exo)can also play the role of myocardial protection by anti-apoptosis,promoting cell survival,promoting angiogenesis and reducing myocardial fibrosis,and its secreted exosomes show more powerful functions than adult tissue stem cell exosomes.At the same time,it was found that the therapeutic effect was mainly mediated by paracrine factors(such as miRNA,etc.).However,the research on iPSC-Exo is still in its infancy,and the specific mechanism of its role is not clear.Therefore,in order to further study the function of iPSC-Exo in the repair of myocardial injury,we extracted iPSC-Exo and the same strain of human foreskin fibroblast exosome(HFF-Exo).Firstly,to explore the functional differences of exosomes in the repair of myocardial injury between the two groups.Secondly,through the analysis of miRNA histochemistry and bioinformatics,to explore the miRNA of significant difference between the two groups,so as to provide theoretical basis for clarifying the mechanism of iPSC-Exo in the treatment of myocardial infarction.[Methods:]The first part:the exosomes were extracted from iPSC and HFF serum-free medium supernatant by sucrose density gradient centrifugation;then,the exosomes were identified by transmission electron microscopy,nanoparticle tracking technology and Western blot;Then,CCK8,cell scratch,Transwell,Western blot and TUNEL apoptosis staining were used to compare the effects of exosomes on the proliferation,migration and apoptosis of myocardial cell H9C2,and the effects of Matrigel like structure formation on HUVEC angiogenesis;Finally,the phagocytosis of exosomes was carried out to verify that exosomes could be phagocytized by target cells to play a role.The second part:randomly select three groups of iPSC-Exo and HFF-Exo,extract the total RNA of exosomes of each group and conduct quality inspection,use high-throughput sequencing technology to sequence the sample miRNA group,analyze the sequenced data,find the significant difference expression miRNA and its main regulatory pathway between iPSC-Exo group and HFF-Exo control group,and explore the key difference through bioinformatics analysis;At last,the key differential miRNAs were verified and analyzed by miRNA histochemistry and re-sequencing.[Results:]The first part:iPSC-Exo and HFF-Exo were successfully separated by sucrose density gradient centrifugation,which were consistent with the characteristics of exosomes after transmission electron microscopy observation,nanoparticle tracking technology and Western blot identification,and could be used for subsequent experiments;CCK8 showed that iPSC-Exo could promote the survival of H9C2 cardiomyocyte injury model induced by H2O2 more than HFF exo,but the effect of both on the proliferation of normal H9C2 cells was not obvious;Western blot was used to detect Cleaved caspase-3 expression,and the results showed that iPSC-Exo could reduce H9C2 induced by H2O2 more than HFF-Exo Cardiomyocyte apoptosis and TUNEL apoptosis staining also showed the same results.Cell scratch and Transwell experiments showed that iPSC-Exo could promote H9C2 cell migration more than HFF-Exo intervention.Matrigel tube like structure formation experiment showed that iPSC-Exo could promote HUVEC angiogenesis more than HFF-Exo intervention.PKH67 was labeled by fluorescence When iPSC-Exo and HFF-Exo were added to H9C2 cells for incubation,it showed that both iPSC-Exo and HFF-Exo could be absorbed into H9C2 cells.The second part:in the iPSC-Exo group and the HFF-Exo control group,the miRNAs were screened by high-throughput sequencing of miRNAs.With P<0.05,fold change>1 as the significant difference,70 miRNAs with significant difference were screened out.Compared with the HFF-Exo group,10 miRNAs in the iPSC-Exo group were up-regulated and 60 were down regulated;70 miRNAs with significant difference were screened Among them,8 gene clusters were found,including 19 miRNAs with significant differences,one of which was up-regulated and seven of which was down-regulated;through go analysis and pathway analysis of target genes regulated by miRNA gene clusters,it was found that they were mainly involved in cell survival,apoptosis,cell migration,angiogenesis and tumorigenesis.Further significant differences in miRNA Through pathway analysis,we found that they regulate four most likely related pathways involved in myocardial injury repair,which are PI3K/Akt signaling pathway,NF-?B signaling pathway,AMPK signaling pathway and JAK/STAT signaling pathway respectively.Finally,we focused on the selection of the most target genes in the regulatory signaling pathway and their combination in myocardial injury repair,which may play an anti apoptosis,promote cell survival and angiogenesis role miRNA with other functions:there are up-regulated miR-92a-3p,miR-103a-3p,miR-106a-5p,down regulated miR-27b-3p,miR-199a-5p,miR-143-3p,miR-221-3p.The analysis of miRNA expression also shows that the above miRNA expression is consistent with the above sequencing results.Therefore,we speculate that these miRNAs carried by iPSC-Exo may be involved in the process of myocardial injury repair.[Conclusion:]1.Compared with HFF-Exo,iPSC-Exo is more significant in promoting myocardial cell viability,anti-apoptosis,promoting myocardial cell migration and promoting endothelial cell angiogenesis.2.Through high-throughput sequencing,there were significant differences in miRNA expression profiles between iPSC-Exo and HFF-Exo control groups.A total of 70 miRNAs with significant differences were screened,10 of which were up-regulated and 60 were down regulated.These miRNAs may participate in the repair of myocardial injury through the four signal pathways of PI3K/Akt,NF-?B,AMPK and JAK/STAT involved in the regulation of target genes.3.MiR-92a-3p,miR-103a-3p,miR-106a-5p,miR-27b-3p,miR-199a-5p,miR-143-3p,miR-221-3p may be related to the role of iPSC-Exo in the repair of myocardial injury.