Experimental Research About The Effect Of Overexpression Of Microrna-206on The Expression Of Hippocampal Neuronal BDNF Induced By Ketamine

Backgroud and ObjectivePrevious studies indicated that the antidepressant effects of ketamine wereinvolved in the expression of neuronal BDNF, but the underlying mechanismwas still unclear. Researches demonstrated that microRNAs(miRNAs) couldregulate the expression of the target gene mRNA through posttranscription level,and miRNA-206(miR-206) could downregulate the expression of neuronalBDNF. Our previous studies in vivo and vitro showed that ketamine couldsignificantly reduce the expression of the miR-206, and increase the expressionof BDNF. The main aim of the study is to investigate the effects ofoverexpression of miR-206on the relative miR-206and BDNF expressionlevels and cell function of hippocampal neurons treated by ketamine.MethodsPrimary hippocampal neurons are cultured for7days and randomly dividedinto four groups, including control group, transfection group, ketamine groupand transfection plus ketamine group, and repeated three times/timepoint/group. Control group: hippocampal neurons were cultured with normalcultured medium for6and48h; Transfection group: hippocampal neuronscontinued to be cultured with normal cultured medium for6and48h at4h after hippocampal neurons were transfected with pre-miR-206; Ketamine group:hippocampal neurons continued to be cultured with normal cultured mediumfor6and48h at6h after hippocampal neurons were treated with100μMketamine (final concentration); Transfection plus ketamine group: hippocampalneurons continued to be cultured with normal cultured medium for6and48h at6h after hippocampal neurons transfected with pre-miR-206for4h were treatedwith100μM ketamine (final concentration). RNA was extracted for miR-206analysis with qRT-PCR technique at6h after hippocampal neurons of eachgroup were finally treated. At48h after hippocampal neurons of each groupwere finally treated:①hippocampal neuronal apoptosis were analyzed withTUNEL;②The relative BDNF protein expression levels were analyzed withwestern blot technique.③The voltage activated calcium currents(Ica(HVA)) andthe transient outward potassium current(IA) of single hippocampal neuronal cellwere recorded with patch clamp technique.④The longest neurites neurons(LPD), number of branches (PDs) and Total length of neurites (TDL) wereobserved under the microscope.ResultsFactorial design ANVOA revealed that the relative miR-206and BDNFexpression levels, the apoptosis rates and Ica(HVA)or IAof hippocampalneurons were significantly influenced by not only the two factors of miR-206transfection and ketamine (p<0.01) but also their interaction (p<0.01). Furtheranalysis by one way ANVOA and LSD’s test, we found: Compared with control group:①the relative miR-206levels and value of Ica(HVA)or IAin ketaminegroup were obviously decreased (p<0.01), and the relative BDNF proteinlevels were significantly increased (p<0.01), however the apoptosis rate had nostastic difference between ketamine and control group;②the relative miR-206levels, the apoptosis rate and value of Ica(HVA)or IAin the transfection groupwere significantly increased (p<0.01), however the relative BDNF proteinlevels were significantly decreased (p<0.01);③the relative miR-206levels, theapoptosis rate and value of Ica(HVA)or IAhad no stastic differences between thetransfection plus ketamine and control group（P>0.05）. Compared withketamine group:①the relative miR-206levels, the apoptosis rate and value ofIca(HVA)or IAwere obviously increased in the transfection group (p<0.01),however the relative BDNF protein levels were significantly decreased(p<0.01);②the relative miR-206levels and value of Ica(HVA)or IAin thetransfection plus ketamine group were significantly increased (p<0.01), and therelative BDNF protein levels were significantly decreased (p<0.01), howeverthe apoptosis rate had no stastic difference between ketamine and transfectionplus ketamine group (P>0.05). Observation of LPD, PDs and TDL of neuronsunder microscope showed no statistical difference among the four groups (P>0.05).Conclusions1、The overexpression of miR-206in hippocampal neurons could obviouslyinhibit the BDNF protein expression induced by ketamine. 2、The neuronal apoptosis may be related to the high Ica(HVA)or IAinduced bythe overexpression of miR-206.