| [Objective] In earlier days study, we investigate microRNAs which specially expressed in transected spinal cord of rats by microRNA microarray analysis.In this study miR-31 was one of which was selected and further to screen the target gene of miR-31 in nervous system. We investigate the change of relevant target genes mRNA in vitro by miR-31 regulation and analyzes the mechanism.According to the study could provide important evidence for the mechanism of regulation by miR-31 after spinal cord injuries.[Materials and Methods] In earlier days study, through screening the analysis result, we obtained microRNAs which specially expressed in transected spinal cord of rats, miR-31 as one of which was selected in this study. Then we utilized bioinformatics to analyzed target genes which were predicted of miR-31, combined with the previous study in our laboratory and reports of literature to further screen as candidated target molecule. Then miR-31 sequences were transfected into PC12 cells in vitro, which divided into 3 groups respectively, were normal cells group, group only transfected liposome and miR-31 transfected group, we investigated the effects of miR-31 transfection on cellular morphology, numbers and activity of cells cultured in vitro.The target gene in each group were detected in mRNA by RT-PCR and the proteins level of target molecule were detected by immunocytochemistry image analytical technique. The data was statistical treated with one way ANOVA.[Results]1. We have slected 6 target genes combine with RT-PCR that were predicted regulated by miR-31, which were eukaryotic translation initiation factor 5 (EIF5), BRCA1 associated protein-1 (ubiquitin carboxy-terminal hydrolase) (BAP1) syntaxin 12 (STX12),VAMP (vesicle-associated membrane protein)-associated protein B and C (VAPB),Calciumchannel,voltage-dependent, beta 2 subunit (CACNB2) and regulator of G-protein signalling 4 (RGS4)2. By liposome-mediated miR-31 Transfection of PC 12 cells, the optimal concentration of miR 31 (pmol):liposome (μl) is equal to 20:1. After transfection of miR-31 in vitro, number of PC 12 cells were statistically decreased.The activity and shape of PC12 cells were no statistically changed in three group. It is indicated that PC 12 cell proliferation was restrained by miR-31.3. The mRNA of target factors were detected by RT-PCR. Among CACNB2 and RGS4 mRNA were not detected in PC 12 cells. EIF5,BAP1, STX12 and VAPBmRNA all were expressed in PC 12 cells. It were no statistically changed in liposome transfected group compared with normal cells group. But EIF5 (p=0.048,p<0.05)and BAP1(p=0.008,p<0.01) mRNA were statistically raised in miR-31 transfected group compared with only liposome transfected group in PC 12 cells. To compare BAP1 and EIF5, STX12 mRNA were statistically reduced in miR-31 transfected group, compared with only liposome transfected group in PC 12 cells (p=0.007, p<0.01) VAPB mRNA was not statistically changed in miR-31 transfected group.4. EIF-5 and BAP1 have postive expressed in normal spinal cord of rat by immunocytochemistry.EIF-5 was distributed in endochylema of neuron and gliocyte. BAP1 stain fairly lighter. It was distributed in endochylema of neurons,particularly dorsal horn neurons; Immunocytochemical stain display that EIF-5and BAP1 were expressed in PC 12 cells. The immunopositive product was locatde in cytolymph. but the OD value of all performed no statistical significance among miR-31 transfected group.[Conclusions]1. In experiment of PC 12 cell line confirm that miR-31 induce apoptosis of PC 12 cells and STX12 was the direct target factor of miR-31. STX12 mRNA expression level was down-regulated by miR-31.2. BAP1 and EIF5 mRNA expression level possiblly were up-regulated by miR-31 indirect regulation.It is mechanism unknown. 3. In this study, VAPB mRNA was not regulated by miR-31...
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