Electrical Stimulation Promotes The Migration Of Schwann Cells Via Inducing MiR-741-3p To Regulate Radil Expression

Objective:To investigate the effect of electrical stimulation-induced miR-741-3p expression and its regulated Radil on the migration of Schwann cells(SCs).Methods:1.A total of 12 adult male Sprague-Dawley rats(180-220g)were randomly divided into sciatic nerve crush injury + ES group and sciatic nerve crush injury group(control group),with 6 rats in each group.Rat model of sciatic nerve crush injury was established in two groups.At 7 days after surgery,rats were sacrificed.The Injured nerve were taken and used for analysis.The difference in miR-741-3p expression between the ES and control groups was detected using fluorescence in situ hybridization assay.2.The target genes of miR-741-3p were predicted by three databases,including Target Scan,miRWalk and miRDB.3.Verification of the targeting relationship between miR-741-3p and Radil in Schwann cell migration.(1)The effect of miR-741-3p on the migration of SCs.SCs were transfected with miR-741-3p mimic,miR-741-3p inhibitor and miR-741-3p mimic-con,miR-741-3p inhibitor-con.(2)The effect of Radil on the migration of SCs.Radil-small interfering RNA(siRNA)and control siRNA were transfected into SCs,migration ability of SCs after transfection were detected by Transwell assay.(3)Validation that miR-741-3p regulates SCs migration by targeting Radil.The experiment was divided into two groups,one group transfected the miR-741-3p inhibitor and miR-741-3p inhibitor-con into SCs,and the other group transfected the miR-741-3p mimic and miR-741-3p mimic-con into SCs in which the migration ability of SCs was detected by Transwell,while the expression of Radil was detected by RT-PCR.(4)Rescue assay: miR-741-3p inhibitor+siRNA and miR-741-3p inhibitor +control siRNA were transfected into SCs,the changes in migration ability of SCs were detected by Transwell assay.miR-741-3p mimic+Radil plasmids and miR-741-3p mimic+control Radil plasmids were transfected into SCs,the changes in migration ability of SCs were detected by Transwell assay.Results:1.The mean intensity of green fluorescence analyzed using Image J software was 13±5 in the ES group,and 34±11 in the control group,there were statistically significant differences between the two groups(P<0.05).These results suggest that ES could down-regulate miR-741-3p expression at7 days after sciatic nerve injury.2.Using Target Scan,miRTar Base,and miRDB databases to predict the target genes,a total of 69 genes were predicted as possible target genes of miR-741-3p.Among these target genes,Radil gene mainly mediated cell migration and adhesion.3.ES promotes the migration of SCs via down-regulating miR-741-3p to increase the expression of its target gene Radil.(1)The number of migrated SCs in the miR-741-3p inhibitor group was significantly higher than that in the NC group(156±48 vs.105±37,P<0.01),whereas the number of migrated cells in the miR-741-3p mimic group was significantly lower when compared to NC group(86±26 vs.138±59,P<0.01),suggesting that miR-741-3p inhibits the migration of SCs in vitro.(2)There were statistically significant differences in number of migrated SCs between the Radil-siRNA and control siRNA groups(95±12 vs.137±33,P<0.01).Since Radil siRNA down-regulates the expression of Radil,the results suggest that Radil has an important role in the migration of SCs.(3)The results of the RT-PCR assay showed that the m RNA expression level of Radil was significantly higher in the miR-741-3p inhibitor group than that in the NC group and the migratory ability of Schwann cells increased(148±41vs.94±12,P<0.05);the m RNA expression level of Radil was significantly lower in the miR-741-3p mimic group than that in the NC group and the migratory ability of Schwann cells reduced(68±7vs.103±9,P<0.05).It shows that miR-741-3p may affect the migration of Schwann cells by regulating the expression of Radil.(4)There were statistically significant differences in number of migrated SCs between the miR-741-3p inhibitor + siRNA and miR-741-3p inhibitor +control siRNA groups(87±19 vs.133±25,P<0.01),between the miR-741-3p mimic + plasmid and miR-741-3p mimic + control plasmid groups(118±14vs.84±11,P<0.01).The results suggest that Radil knockdown or overexpression could reverse the promotion effect of miR-741-3p inhibitor or the inhibition effect of miR-741-3p mimic the migration of SCs,which further confirm that miR-741-3p promotes the migration of Schwann cells via regulating Radil expression.Conclusions:The ES promotes Migration of Schwann cells through down-regulating miR-741-3p to up-regulate the expression of its target gene Radil.