Construction Of MiRNA-21-5P-loaded Engineering Exosomes And Its Effect And Mechanism On DRG Neurons

The repairment after peripheral nerve injury is a very important topic in clinical research.Peripheral nerves have certain regenerative capacity.The activation of relavant transcription factors and the regulation of regeneration-related genes are the keys to the survival of the neurons,as well as their axonal regeneration and functional recovery.Micro RNA(miRNA)and exosomes play an important role in this process.miRNA can regulate the expression of genes.By targeting the 3’-untranslated region(3’-UTR)of the target mRNA molecules,miRNA inhibits the translation or degradation of mRNA and negatively regulates the expression of related genes.A variety of miRNAs are closely related to genes that are involved in regulating regeneration after nerve injury.Exosomes(Exo)are natural nano-scale drug carriers,which can mediate the transfer of RNAs and other signalling molecules between cells,as well as bring genetic material into target cells,and thereafter regulate the expression of the related genes.Objective:To Construct miRNA-21-5P-loaded engineering exosomes,and to explore the feasibility and mechanism of using them to promote the regeneration of DRG neurons.It provides experimental research basis for effective manipulation of nerve regeneration related genes to promote peripheral nerve injury repairment.Methods:1.Isolation and extraction of bone marrow mesenchymal stem cells(BMSCs)-derived exosomes.2.Identification of exosomes by transmission electron microscopy.3.Nanoparticle Analyzer(NTA)measured the size,concentration and zeta potential of exosomes.4.To load exogenous miR-21-5p/ miR-NC into exosomes by electroporation,in order to construct engineering exosomes.5.The miRNA introduction efficiency was measured by Apogee Micro flow cytometer.6.DRG neurons of SD rats were cultured in vitro.iExosomes-21,iExosomes-NC,Exosomes,miR-21-5p plus Exosomes(miR-21-5p+ Exo),miR-21-5p plus transfection reagent(Lipofectamine3000;miR-21-5p + LP),and PBS were added respectively7.Using PKH67/Di I to label exosomes or engineered exosomes,and FAM/CY3 to label miRNA-21-5p,and then co-culture them with ex vivo DRG cells,in order to observe neurons and glial cells after adding the exosomes and miRNAs.8.The activity of ATPase was detected by Cell Titer-Glo? and the effect of each group on the activity of DRG cells in vitro was observed.9.To detect the activity of apoptosis-related protease Caspase3/7 by Caspase-Glo?3/7,and to observe the effect of miRNA-21-5p on the expression of Caspase3/7.10.Hoechst 33342 /PI double staining was used to detect the apoptosis and necrosis of each group,and observe the protective effect on neurons.11.The mRNA and protein expression levels of miRNA-21-5p target genes Spry,TIMP3 were detected by RT-PCR and Western blot,in order to verify the effect of miRNA-21-5p on the regulation of related target genes.12.To detect the effect of each group on neurite outgrowth by cellular immunofluorescence.Results:1.The exosomes derived from BMSCs were successfully isolated and identified by TEM and NTA.2.When using 400 v,2.5 ms/15 ms electroporation to introduce miR-21-5p into exosomes,the loading efficiency in the 15 ms group was 4.78%,which was significantly higher than 1.15% in the 2.5 ms group(P <0.01).3.Exo and miRNA derived from BMSCs could be taken up by neurons and glial cells.4.Compared with PBS group,the ATPase activity of mir-21-5p + LP group decreased significantly(P < 0.001)at 24 h in each group.5.After 72 hours of culture in each group,there was no significant difference in the expression of Caspase3/7 between the groups(p>0.05).6.After 24 hours of culture in each group,the number of apoptotic necrosis in the iexosomes-21 group was significantly lower than that of the other groups(p<0.01),and the number of apoptotic necrosis in the miR-21-5p+LP group and the PBS group was higher than that of the other groups(P <0.01).7.The iexosomes-21 can effectively reduce the levels of TIMP3 mRNA,Spry2 mRNA and Spry2 protein(p <0.001).8.The length and number of neurons in the iExosomes-21 group and the miR-21-5p +LP group were significantly larger than those in other groups(p <0.001).Conclusions:Engineering exosomes can be used to transfer exogenous miRNA-21-5p into DRG cells in vitro,which can protect neurons by promoting the growth of neuronal processes and inhibiting apoptosis.The mechanism may be related to down-regulate the expression levels of TIMP3 and Spry2,but not the expression levels of caspase3/7.Engineering exosomes may provide a new strategy for the treatment of nerve injury through promoting nerve repairment.