The Post-transcriptional Regulation Role Of Mi R-1 In Peripheral Nerve Regeneration By Targeting BDNF

Objective: Nerve damage, especially peripheral nerve injures are quite common in clinical practice, and the incidence rate shows a significant increase. After traumatic injury, peripheral nerves can spontaneously regenerate, but for some cases of serious neurological injures, the nerve repair and anastomosis technique still exist some limitations and the treatment effect is far from expectation. The rat sciatic nerve is a classical model of the study of peripheral nerve regeneration. The expression level of many genes is changed in the proximal nerve during sciatic nerve injury and regeneration process, indicating that the expression of these genes is subject to certain regulatory. micro RNA(mi RNA), a kind of small non-coding RNA containing about 22 nucleotides, can regulate gene expression at post-transcriptianl level, providing a new mode of gene regulation. Brain-derived neurotrophic factor(BDNF) plays an important role in the regulation of peripheral nerve regeneration.This thesis aims at exploring the post-transcriptional regulation role of mi R-1 during peripheral regeneration by targeting BDNF.Methods: According to our preliminary laboratory results of microarray analysis, BDNF showed a negative correlation between mi R-1 in the sciatic nerve regeneration process. mi R-1 may target and regulate BDNF 3’-UTR according to the prediction through the software of Target Scan and Mi Randa, and there are three mi R-1 target sites in the BDNF 3’-UTR. BDNF 3’-UTR and BDNF 3’-UTR three kinds of mutants were subcloned into luciferase reporter vector to generate p-Luc-UTR reporter plasmid. We then preformed dual-luciferase reporter assay to determine whehter mi R-1 can directly target the BDNF 3’-UTR, and the importance of the three target sites. Total RNA and proteins were extracted from the SCs which transfected with mi R-1 mimetics and inhibitors. Real-time PCR and Western blotting analysis to determine the mechanism of mi R-1 regulate BDNF in the post-transcriptional level. Then forty adult, male Sprague-Dawley rats(180-220 g) were provided by the Experimental Animal Center of Nantong University and randomly divided into five groups(eight rats per group) to construct the rat left hind sciatic nerve transection model. One group of rats was euthanized immediately as control group without sciatic nerve resection(0 h), and the other groups were euthanized and 0.5 cm of the proximal sciatic nerve was collected at 1, 4, 7, and 14 d post nerve transaction respectively. Then we performed Real-time PCR to analyze the expressiong level of mi R-1 and BDNF m RNA at different time points after sciatic nerve injury in the proximal nerve; Western blotting to analyze the expressiong level of BDNF protein. Then the proliferation and migration of SCs were assayed by Cell-Light? Ed U Apollo In Vitro Kit and Transwell, respectively. The secretion of BDNF in SCs were analyzed by Chemi Kine? BDNF Sandwish ELISA Kit. And then we determine mi R-1 regulate the proliferation and migration of SCs by targeting BDNF though the rescue experiments.Results: mi R-1 can be targeted BDNF 3’-UTR directly, and the three mi R-1 target sites in BDNF 3’-UTR are equally important. mi R-1 suppress the expression of BDNF via the mechanism of degradation of BDNF m RNA. BDNF demonstrated negatively correlated expressions with mi R-1 at 0, 1, 4, 7 and 14 d in the sciatic nerve regeneration process, providing further evidence for regulation of BDNF by mi R-1 at the post-transcriptional level. mi R-1 inhibited cell proliferation, migration of primary SCs through directly targeting BDNF, and decreased the secretion of BDNF in SCs.Conclusions: This study, for the first time, revealed that BDNF expression was regulated by mi R-1 at the post-transcriptional level during peripheral nerve injury and regeneration. Further, we discovered that mi R-1 could inhibit proliferation and migration of SCs by targeting BDNF, and decrease the secretion of BDNF in SCs. Collectively, our data first revealed that mi R-1 played a dynamic regulatry role in peripheral nerve regeneration. Since the fragment of mi RNA is small enough to pass through the blood nerve barrier, the regulating of BDNF by mi R-1 in nervous system disorders will avoid the limitations of BDNF in the clinical application, which enriching the study of regulatory roles of mi RNA and contributing to the development of novel therapeutic targets.