Study Of MiR-132 Regulates BDNF-TrkB Signaling Expression In Epileptic Neurons

Objective:Epilepsy is a kind of common intractable epilepsy in adults and its pathogenesis has not been fully understood. BDNF(brain derived neurotrophic factor) has the effect of neuronal survival, differentiation and growth. It achieves its function when combines with Trk B(tyrosine kinase receptor B) and activates BDNF-Trk B signaling which is consided mportant in epileptogenesis. Micro RNAs(mi RNAs) are an important class of noncoding RNA, which regulate gene expression at the post-transcriptional level. They have now been confirmed that it plays an important role in a variety of diseases of the nervous system. Studies have shown that mi R-132 plays an important role in the process of epilepsy, but specific mechanisms need to be researched.To explore the functions of mi R-132 in BDNF-Trk B signaling and in the pathogenesis of TLE, we observed the dynamic change of p-Trk B( phosphorylated Trk B) in epilepsy model of cultured hippocampal neurons of rats after transfected mi R-132.Method: In this study, mi R-132 lentivirus vector was constructed.Primary hippocampal neurons prepared from new born SD rats were cultured in vitro, and 7 days later. The cells were randomly divided into ①normal group、②epilepsy group、③normal+BDNF group、④epilepsy+BDNF group、⑤normal+mi R-132 group、⑥epilepsy+mi R-132 group、⑦normal+mi R-132+BDNF group and ⑧epilepsy+mi R-132+BDNF group.Using the patch clamp technique detect hippocampal neuron discharge conditions.Using QPCR method to detect the expression of mi R-132 in hippocampal neurons. Using western blotting technique to detect Trk B and p- Trk B protein expression in hippocampal neurons.Gray value was analyzed by quantity one software. Relative gray values of p-trk B/trk B protein indicated activation of BDNF-Trk B signaling and were statistically analyzed using analysis of variance. p≤0.05 means statistically significant difference.Epilepsy model of hippocampal neurons were established by being exposed to Mg2+ free media for 3 hours.Mi R-132 transfection groups were cultured with mi R-132 lentivirus diluent for 12-24 hours on the 7th day and then returned to normal media for 24 hours. BDNF was added into media 10 min before cell collection.Result: 1、The patch clamp technique epilepsy detect hippocampal neuron discharge: Compared with normal group, the spontaneous discharge frequency in the epilepsy group is obviously increased(p=0.001).2、QPCR method to detect the expression of mi R-132 :Compared with the normal group, the mi R-132 expression in epilepsy group was obviously increased, the difference was statistically significant(p<0.001).3、Western Blot results:Compared with epilepsy group,the p-Trk B/Trk B value in control group was higher(p=0.008).Compared with control group, the p-Trk B/Trk B value in control+BDNF group was higher(p<0.015). Compared with epilepsy group,the p-Trk B/Trk B value In epilepsy+BDNF group was higher(p=0.012). Compared with epilepsy+mi R-132 group,The p-Trk B/Trk B value in epilepsy group and epilepsy+BDNF+mi R-132 group is higher(p=0.004;p=0.030);Conclusion: 1、In the condition of epilepsy, the expression of mi R-132 in hippocampal neurons is increased.2、In vitro cultured hippocampal neurons, p-Trk B/Trk B value was significantly elevated after adding BDNF both under normal condition and epileptic condition. It proved that BDNF-Trk B signaling can be activated by exogenous BDNF.3、The p-Trk B/Trk B value under epileptic condition was lower than control group.It proved that BDNF-Trk B signaling is inhibited under epileptic condition.4、The p-TrkB/TrkB value under epileptic condition was lower when mi R-132 was transfected.It indicated that the transfection of mi R-132 inhibited BDNF-Trk B signaling.