Abstract:

Objective To discuss the effect of microRNA-34a-5p （miR-34a-5p） on the neuron apoptosis in hippocampus tissue of the rats with temporal lobe epilepsy， and to clarify its mechanism. Methods Fifty-two male SD rats were randomly divided into control group， model group， miR-34a-5p inhibitor group， and inhibitor negative control group， and there were 13 rats in each group. The PONEMAH 6.X experimental animal telemetry platform was used to record the electroencephalogram （EEG） of the rats in various groups； real-time fluorescence quantitative PCR （RT-qPCR） was used to detect the expression levels of miR-34a-5p in hippocampus tissue of the rats in various groups； HE staining was used to observe the morphology of hippocampus tissue of the rats in various groups； TUNEL method was used to detect the apoptotic rates of neurons in hippocampus tissue of the rats in various groups； immunohistochemistry method was used to determine the positive expression rates of CDK6， p-Rb， and E2F1 proteins in hippocampus tissue of the rats in various group； Western blotting method was used to detect the expression levels of cyclin-dependent protein kinase 6 （CDK6）， phosphorylated retinoblastoma protein （p-Rb）， and E2F transcription factor 1 （E2F1） proteins in hippocampus tissue of the rats in various groups. Results No abnormalities were observed in the rats in control group； the rats in model group， miR-34a-5p inhibitor group， and inhibitor negative control group exhibited varying degrees of drooling， trembling， bloody tears， staring， chewing tremors， followed by nodding and blinking， and finally forelimb convulsions， standing upright， and falling. Compared with control group， the total duration of epileptic seizures of the rats in model group was significantly prolonged （P<0.01）； compared with model group， the total duration of epileptic seizures of the rats in miR-34a-5p inhibitor group was shortened （P<0.01）； compared with miR-34a-5p inhibitor group， the total duration of epileptic seizures of the rats in inhibitor negative control group was prolonged （P<0.01）. The RT-qPCR results showed that compared with control group， the expression level of miR-34a-5p in hippocampus tissue of the rats in model group was increased （P<0.01）； compared with model group， the expression level of miR-34a-5p in hippocampus tissue of the rats in miR-34a-5p inhibitor group was increased （P<0.05）； compared with miR-34a-5p inhibitor group， the expression level of miR-34a-5p in hippocampus tissue of the rats in inhibitor negative control group was increased （P<0.01）. The HE staining results showed that compared with control group， the cell arrangement in model group was disordered； compared with model group， the cell arrangement in miR-34a-5p inhibitor group was orderly； compared with miR-34a-5p inhibitor group， the cell morphology in inhibitor negative control group was irregular. The TUNEL staining results showed that compared with control group， the apoptotic rate of neurons in CA1 region of hippocampus tissue of the rats in model group was increased （P<0.01）； compared with model group， the apoptotic rate of neurons in CA1 region of hippocampus tissue of the rats in miR-34a-5p inhibitor group was decreased （P<0.05）； compared with miR-34a-5p inhibitor group， the apoptotic rate of neurons in CA1 region of hippocampus tissue of the rats in inhibitor negative control group was increased （P<0.05）. The immunohistochemistry results showed that compared with control group， the positive expression rates of CDK6， p-Rb and E2F1 proteins in hippocampus tissue of the rats model group were increased （P<0.05 or P<0.01）； compared with model group， the positive expression rates of CDK6， p-Rb and E2F1 proteins in hippocampus tissue of the rats in miR-34a-5p inhibitor group were decreased （P<0.05）； compared with miR-34a-5p inhibitor group， the positive expression rates of CDK6， p-Rb and E2F1 proteins in hippocampus tissue of the rats in inhibitor negative group were increased （P<0.05 or P<0.01）.The Western blotting results showed that compared with control group， the expression levels of CDK6， p-Rb， and E2F1 proteins in hippocampus tissue of the rats in model group were increased （P<0.01）； compared with model group， the expression levels of CDK6， p-Rb， and E2F1 proteins in hippocampus tissue of the rats in miR-34a-5p inhibitor group were decreased （P<0.05 or P<0.01）； compared with miR-34a-5p antagomir group， the expression levels of CDK6， p-Rb， and E2F1 proteins in hippocampus tissue of the rats in inhibitor negative control group were increased （P<0.05 or P<0.01）. Conclusion The expression of miR-34a-5p is upregulated in the hippocampal tissue of temporal lobe epilepsy rats， and hippocampal neuron apoptosis is increased. Inhibition of miR-34a-5p expression can reduce the hippocampal neuron apoptotic rate， and its mechanism may be related to the regulation of CDK6， p-Rb， and E2F1 protein expressions in the hippocampus tissue by miR-34a-5p.

Key words: MicroRNA-34a-5p, Epilepsy, Apoptosis, Cyclin-dependent protein kinase 6, Phosphorylated retinoblastoma protein, E2F transcription factor 1