| Objective This study aimed to explore the differential expression micro RNAs(miRNAs)after spinal cord injury(SCI),and further studying the role of key miRNAs in neuronal axon regeneration.Methods Rat sciatic nerve conditioning injury(SNCI)model,simple spinal dorsal column injury(SDCL)model and control group model(without spinal cord injury)were established respectively.Differentially expressed miRNAs in dorsal root ganglion(DRG)neurons were screened according to the microarray.Subsequently,q RT?PCR was performed to validate the expression profiles of miRNAs and measure the expression level of key miRNA in different groups.The target gene of key miRNA was confirmed from the Target Scan database and Luciferase Reporter Assay.Further,based on Immunocytochemistry(IHC)the mean total neurite length(per neuron)of DRG neuronal axons labeled by NF-200 was analyzed and measured in different groups after inhibiting the expression of key miRNA.Meanwhile,Western Blot analysis was performed to investigate the expression of P27,Growth-associated protein 43(GAP-43),Acetyltransferase of α-tubulin(α-TAT1)and a-tubulin proteins.IHC was performed to investigate the relative fluorescence intensity expression of P27,GAP-43 and atubulin proteins.Chondroitin sulfate proteoglycans(CSPGs)were added to the medium of DRG neurons in different groups to form inhibitory microenvironment of axon growth,and then to investigate the expression of P27,GAP-43,α-TAT1 and a-tubulin proteins and the mean total neurite length(per neuron)again.Results The expression profiles of miRNAs revealed that 681 miRNAs were differentially expressed at least once in the adult rat DRGs,and there are 47 miRNAs expression multiples of changed more than 1.2 times at each time point,the expression of miR-221-3p has significant difference among all the altered miRNAs.q RT?PCR indicated that the expression of miR-221-3p was down-regulated in the SNCI group but up-regulated in the SDCL group,and the miR-221-3p expression at the same four checkpoints was consistent with the microarray expression trend.Subsequently,Target Scan database and Luciferase Reporter Assay confirmed that p27 was the target gene of miR-221-3p.Further,IHC indicated that the mean total neurite length was significantly longer in the miR-221-3p inhibitor group,also the relative fluorescence intensity of P27,GAP-43 and α-tubulin was up-regulated.Meanwhile,Western Blot analysis showed that p27,GAP-43,α-TAT1 and α-tubulin expression was up-regulated in the miR-221-3p inhibitor group.Still,after CSPGs were used in this study,the results demonstrated that the inhibition of miR-221-3p also could enhance p27,GAP-43,α-TAT1 and α-tubulin expression and boost neuronal axon regeneration.Conclusion1.Based on the expression profiles miR-221-3p probably played a pivotal role in promoting axonal regeneration after SCI.2.Inhibition of miR-221-3p expression could enhance p27,GAP-43,α-TAT1,and α-tubulin expression and advance axonal growth in DRG neurons.3.In the inhibitory environment with CSPGs,miR-221-3p also could contribute significantly to the regeneration of DRG neurons by specifically regulating p27 in the p27/CDK2/GAP-43 and p27/α-TAT1/α-tubulin pathways. |
