Study of miRNA-212 regulating BDNF/TrkB signaling pathway in epileptic neuron model

Abstract

Objective To study the effect of epileptic neuron model after transfection of microRNA (miRNA)-212 on brain-derived neurotrophic factor (BDNF)/tyrosine protein kinase B (TrkB) signaling pathway.  Methods The primary rat hippocampal neurons were cultivated in vitro for 7 d and were randomly divided into 8 groups: control group, epilepsy group, control + BDNF group, epilepsy + BDNF group, control + miRNA-212 group, epilepsy + miRNA-212 group, control + miRNA-212 + BDNF group, epilepsy + miRNA-212 + BDNF group. Epilepsy model of hippocampal neurons were established by being exposed to Mg²⁺ free extracellular fluid for 3 h. And then the neurons were put back into the normal extracellular fluid of magnesium for 2 h and slow virus diluent was dropwise added for transfection, so that miRNA-212 lentiviral vector was structured. Protein was extracted after 48-72 h. BDNF was added into media 10 min before protein was extracted. Immunofluoresence double staining, patch clamp technique and Western blotting were used to observe the effect of miRNA-212 transfection on BDNF/TrkB signaling pathway. Results After BDNF was injected, compared with the control group and epilepsy group, the phosphorylated TrkB (pTrkB)/TrkB value was significantly higher in control + BDNF group and epilepsy + BDNF group (P = 0.001), suggesting the BDNF/TrkB signaling pathway was activated. After transfection of miRNA-212, the pTrkB/TrkB value in epilepsy + miRNA-212 + BDNF group was significantly lower than that in epilepsy + BDNF group (P = 0.001), suggesting the BDNF/TrkB signaling pathway was suppressed.  Conclusions When hippocampal neurons were transfected miRNA-212, the BDNF/TrkB signaling pathway was suppressed, but BDNF could activate the BDNF/TrkB signaling pathway.

doi: 10.3969/j.issn.1672-6731.2014.11.010