The Expression Of MiR-124 Was Changed In Transected Spinal Cord Of Rats And Studied The Effects Of MiR-124 On Target Proteins In Cells Cultured And The Its Mechanism Of Action

[Objective]In this study,we investigate microRNAs which specially expressed in transected spinal cord of rats by microRNA microarray analysis,miR-124 was one of which was selected.Then double strands sequences of miR-124 were transfected cells cultured in vitro to confirm target proteins regulated by miR-124.In addition,the study also detected that miR-124 had influences on cells cultured,and analyzed the mechanism of action.Lastly,according to the study could provide important evidence for the mechanism of regulation by miR-124 after spinal cord injuries.[Materials and Methods]SD rats were divided into 2 groups randomly:2 rats subjected to spinal cord transection in T9 level as operated group,and other 2 rats as sham-operated group.The samples of T10 cord segments from all rats and liver from one sham-operated rat were performed by microRNA microarray analysis.Through screening the analysis result,we obtained 4 microRNAs which specially expressed in transected spinal cord of rats,miR-124 as one of which was selected.Then we utilized bioinformatics to analyzed target proteins which were predicted of miR-124, combined with the previous study in our laboratory and reports of literature to determine 4 target proteins as the study contents.Then miR-124 sequences were transfected into PC12 and Hela cells in vitro,which divided into 3 groups respectively, were normal cell group,group only transfected liposome and miR-124 transfected group.The target proteins in each group were detected both in mRNA and protein level by RT-PCR,immunocytochemistry and Western blot analysis.The data was statistical treated with one way ANOVA to make sure which target proteins were regulated by miR-124.Lastly,we also investigated the effects of miR-124 transfection on cellular morphology,numbers and activity of cells cultured in vitro.[Results]1.There were 4 microRNAs specifically expressed in transected spinal cords of rat by microRNA microarray analysis.2.The expression of miR-124 in T10 cord segments of rats subjected to cord transection was down-regulation.3,We have screened 4 target proteins that were predicted regulated by miR-124,which were pyridoxal kinase(PDXK),voltage-dependent anion channel 2(VDAC2),Rho GDP dissociation inhibitor(GDI) alpha(ARHGDIA) and NT-4.4.PDXK showed immunopositive both in PC12 and Hela cells,but the OD value performed no statistical significance among 3 cell groups.However,PDXK were reduced in the transfected groups by Western blot analysis,compared with normal cell group,and in miR-124 transfected group PDXK appeared more reduce.PDXK mRNA was no significant difference among 3 cell groups.VDAC2 show same change only in PC 12 cells.5.After transfection of miR-124 in vitro,number and activity of PC12 cells were statistically decreased,and the activity of Hela cells decreased till to 72h. However,there was no obvious change in shape of PC 12 and Hela cells.[Conclusions]1.miR-124 was specifically expressed in the transected spinal cord of rats.2.PDXK and VDAC2 were regulated by miR-124 in PC12 cells;while PDXK was in Hela cells and the main pathway of regulation was inhibit protein translation.3. VDAC2 was one of reasons which resulted in numbers and activity of cells cultured decreasing.