The Expression Profile Of MicroRNA In Dorsal Root Ganglion After Sciatic Nerve Injury And Preliminary Probe Into Their Function

Objective To study the expression profile of microRNA(miRNA) in dorsal root ganglion(DRG) after sciatic nerve injury and probe into their function.Methods Adult healthy male Spragu-Dawley (SD) rats, aging from60to90days, were used. The right sciatic nerve was transected and the animals survived for three days, then the right (injured) and left (naive) L4-5DRG were collected for extracting total RNA. The miRNA microarray analysis was performed to screen the differential expression of miRNAs. Quantitative real-time polymerase chain reaction(qRT-PCR) and in situ hybridization were used to validate the data of microarray. After prediction of potential miRNA targets by bioinformatics analysis based on the result of microarray and qRT-PCR, the luciferase reporter vectors were constructed to validate the targets of candidate miRNAs. Finally, the function of the candidate miRNAs was studied by quantifying the neurite length after P-tubulin Ⅲ immunofluorescence histochemistry and detecting the expression of Robo2protein in cultured primary neurons after transfection.Results Microarray assay showed that total of21kinds of miRNA were down-regulated three days after sciatic nerve transection. The down-regulation of miR-144, miR-145and miR-214in primary neurons of DRG after sciatic nerve transection were further validated by qRT-PCR and in situ hybridization. The computational predictions of miRNA targets for miR-144, miR-145, and miR-214showed that the3’-untranslated regions (3’-UTR) of sGAPl and Robo2have binding sites for these candidate miRNAs. Using luciferase reporter assay, we found that miR-145and miR-214down-regulated the luciferase activity of vectors containing the3’-UTR of Robo2and srGAP1respectively. In primary neuron cultures, transfection with miR-145inhibited neurite outgrowth and decreased the levels of Robo2protein.Conclusion There was differential expression of miRNAs in DRG neurons after sciatic nerve injury. Of these miRNAs, miR-145and miR-214were significant down-regulated. The miR-145inhibited neurite growth by decreasing the expression of Robo2and miR-214might regulated the expression of srGAPl, suggesting miRNA may mediate peripheral nerve regeneration through Slit-Robo-srGAP cascade.