Correlation Bettween PTPRR,OPN,miRNA-30e And PTE

Objective:To detect the expression of protein tyrosine phosphatase receptor R type?PTPRR??Osteopontin?OPN?and miR-30e-5p in cortical brain tissue of post-traumatic epilepsy?PTE?and to explore the relationship with PTE.Method:1.12 adult rats were randomly distributed into two groups:6 normal control groups and 6 experimental groups.To establish the PTE model,the rats of experimental group were injected with 0.4 mol/L FeCl_2.2.Real-time PCR and Western blot methods were applied to detect the expression levels of PTPRR,OPN,ERK,miR-30e-5p in the cortical brain tissues of normal control group and experimental group.3.Cell experiments were applied to verify the relationship between PTPRR,OPN,ERK and miR-30e-5p.Using cell transfection method to form four groups:rat microglia+Negtive Control,rat microglia+miR-30e-mimics,rat microglia+miR-30e-inhibitor and rat microglia Plasma cells+inhibitor NC.Then at 24h,48h,72h,96h after transfection,two methods of fluorescence microscopy and RT-qPCR were applied to detect the expression of miR-30e-5p to analyze whether the transfection was successful.AT the same time,Real-time PCR was used to detect the expression levels of PTPRR,OPN and ERK in each group.Result:1.The model of PTE was successfully established:the material showed that frontal lobe injection of brain tissue of the experimental group was softened,showing obvious hemosiderosis.At the same time,obvious GFAP positive can be seen under the microscope.EEG showed epileptiform discharge obviously.The average behavioral score reached 4 points,and rats were observed to have performances consistent with seizures such as wet dog-like shaking,twitching of both forelimbs,loss of balance,rigidity and spasticity.2.The result of Rt-PCR:The expression levels of PTPRR,OPN,and ERK mRNA in the experimental group were significantly higher than the normal group?P<0.01?,and the expression level of miR-30e-5p was significantly lower?P<0.01?.3.The result of Western blot:The expression levels of PTPRR,OPN,and ERK protein in the experimental were significantly higher than the normal group?P<0.01?.4.Results of cell experiments:After transfection,miR-30e was extracted at four time points of 24h,48h,72h,and 96h.The result of Rt-PCR showed that four groups of N.C.?N.C.inhibitor?miR-30e mimics and miR-30e inhibitor compare with each other,the difference have no statistical significance?P>0.05?.Observed under the fluorescence microscope,there was no obvious fluorescence signal,but the primer dissolution curve was good.Due to the low transfection efficiency,the CT values detected in the transfected samples have no reference significance.Using cells from N.C.group,the three genes of PTPRR,OPN,and ERK2 were tested twice.And the result showed that the CT values of the internal reference were normal in two tests,none of the three genes were expressed in the first test and in the second test,ERK2 and PTPRR were not expressed,but OPN was expressed.Replace the normal rat brain tissue neurons to verify the primers,all indicators were expressed.Conclusion:1.The expressions of PTPRR,OPN and ERK are up-regulated and miR-30e-5p is down-regulated in the FeCl₂-induced PTE model.The four are likely to be involved in the formation of PTE.2.in the FeCl₂-induced PTE model,miR-30e-5p may target and regulate PTPRR and OPN,and miR-30e-5p inhibits the expression of PTPRR and OPN.3.miR-30e-5p may regulate the ERK pathway through targeting regulate PTPRR and OPN,which play a role in exciting neurons,leading to cell damage or apoptosis,and finally induced PTE.