| Objectives（1）To investigate the expression of miR-219a-5p in model of etoposide-induced or H2O2-induced in vitro neuronal cell death;（2）To investigate the levels of CCNA2 and CACUL1 in etoposide-induced or H2O2-induced in vitro neuronal cell death;（3）Validation of CCNA2 and CACUL1 as direct targets of miR-219a-5p;（4）To explore the mechanism of miR-219a-5p in TBI,in order to provide new ideas and new methods for the diagnosis and treatment of TBI patients.Methods（1）25μmol/L etoposide was used to induce neuronal cell death,and total RNA were collected at 1h,6h,16h,20h,24h after injury,as well as the control group.In addition,neurons were injured with 150μmol/L H2O2,and total RNA were extracted from the injured neurons at 6h,12h,18h,24h as well as the control group.To measure the levels of miR-219a-5p in TBI models at different time points by using quantitative Real-time polymerase chain reaction,respectively.（2）Bioinformatics was used to screen out CCNA2 and CACUL1 as target proteins of miR-219a-5p,and25μmol/L etoposide was used to induce neuronal cell death,and total protein were collected at 1h,6h,16h,20h,24h after injury,as well as the control group.In addition,neurons were injured with 150μmol/L H2O2,and total protein were extracted from the injured neurons at 6h,12h,18h,24h as well as the control group.Western blot was used to analyze the levels of CCNA2 and CACUL1 protein at different time points in the TBI models,respectively.（3）CCNA2 and CACUL1 were verified to be the target proteins of miR-219a-5p by luciferase reporter constructs and overexpression or inhibition of miR-219a-5p.（4）Western blot analysis of the expression of Akt/Foxo3a signaling pathway and p53/bcl-2 signaling pathway.Results（1）Our data demonstrated that the level of miR-219a-5p was upregulated at6 h after etoposide injury（p<0.05）and remained continuously increased until 24 h when compared to the control group.Furthermore,H2O2 treatment causes a similar pattern（p<0.05）.（2）Bioinformatics software analysis showed that CCNA2 and CACUL1 were the target proteins of miR-219a-5p.In model of H2O2-induced in vitro neuronal cell death,protein CCNA2 and CACUL1 were down-regulated at 6h and continuously decreased to 24h.In the H2O2-induced in vitro neuronal cell death,the expression levels of protein CCNA2 and CACUL1 showed a similar trend.（3）Luciferase reporter assays showed that miR-219a-5p could bind to the 3′-UTR of CCNA2 mRNA and 3′-UTR CACUL1 mRNA,respectively.Overexpression or inhibition expression experiments once again proved that CCNA2 and CACUL1 were the target proteins of miR-219a-5p.（4）We further show that,in the neuronal cell injury model,upregulated miR-219a-5p inhibits the expression of CCNA2 and CACUL1 and further regulating Akt/Foxo3a and p53/Bcl-2 signaling pathway respectively,thereby inducing neuronal apoptosis.Conclusions The level of miR-219a-5p was increased in the neuronal cell injury model,and miR-219a-5p negatively regulated the target proteins CCNA2 and CACUL1,thereby regulating the apoptosis of neurons.This experiment suggested that miR-219a-5p may be an important molecule promoting the occurrence and development of TBI,and also provided a new idea for the diaognosis and treatment of TBI. |
