The Different Expression Profile Of MiRNA In Hippocampal Between The Status Epilepticus Rat And Normol Rat

Objective.This study compared the different of miRNA expression in hippocampal region of brain tissue between the Lithium chloride pilocarpine SE model rat and the normal rat brain tissue by using the miRNA microarray chip technology. Futher, the miRNA which showed much more different between the two group were chioced and their expression in the brain tissue and in the peripheral blood were measured by real time PCR. Bio-informatics is used in predicting and examining of the target gene. Discussions also been brought on how the related miRNA injuring the central nervous system of rats after SE.Method1.Setted up model:Origin 6-8 weeks healthy male SD rats, then set up the Lithium chloride-pilocarpine SE model.24 hours after model setting, take the brain tissue and peripheral blood to compare with normal male SD rat's brain tissue.2.Screened the differential expression of miRNA by using miRNA microarray chip technology:extracted miRNAs in the hippocampus of rat brain tissue. Using miRNA microchip technology to study on the miRNA expression difference between hippocampus brain tissue of SE rat and brain tissue of normal rat. 3.Verified the differential expression of miRNA Ration examination by quantitative RT-PCR:Significantly differentially expressing miRNAs were detected by quantitative RT-PCR in hippocampus brain tissue. Also tested the expression in peripheral blood of rat and compared the expression difference between hippocampus brain tissue and peripheral blood.4.Predicted the target genes by bioinformatics analysis:Software of Miranda, TargetScan and PicTar were used to predict the target gene of significantly differentially expressing miRNAs.5.Detected on the target genes using Western Blot:According to the prediction result, using Western Blot inspected the expression of target genes in hippocampus brain tissue of SE rats and normal rats.Result1. Differentially expression miRNA in hippocampus brain tissue of SE rats and normal rats:Using miRNA Microarray chip technology,37 miRNA raise up gene were found in the hippocampus brain tissue of SE rat. Among those, raise up five times higher miRNA gene is miR-34a,miR-22,miR-125a,miR-213,miR-30c,miR-26a,miR-375,miR-99a,miR-24,miR-124a,miR-101-1. miR-34a has the maximum expression difference (25.86 times) while miR-188 has the minimum expression difference (1.42 times). Lower 5 times genes are miR-21,miR-29a,Let-7e,miR-181,miR-215,miR-128a,miR-181b, the miR-21 has the sharpest lowering which is 27.97 times.2.Validated the differentially expressed miRNAs by quantitative RT-PCR:Chose the most obviously 3 increased miRNA (miR-34a,miR-22å’ŒmiR-125a) and 2 decreased (miR-21å’ŒmiR-29a). By using quantitative RT-PCR, the differentially expressed miRNAs in hippocampus brain tissue and peripheral blood were detected and compared. In the five miRNA under test,the miR-34a,miR-125a and miR-22's expression was increased while miR-21 decreased which was consistent with chip test results. Then the result of miR-29a was not. All the target miRNA changing trends in the brain tissue and peripheral blood tissue were consistent. The miR-34a, miR-125a and miR-34a expression in brain tissue was higher than in peripheral blood tissue. And the miR-22 and miR-21 expression in brain tissue was lower than in the peripheral blood tissue.3. Predicted results on target genes:By use of bioinformatics methods, miR-34a, miR-125a, miR-22 and miR-21's target genes were predicted. The results showed that the upregulation of miR-34a and miR-125a could control the expression of Bcl-2 and downturn miR-21 was likely to control the expression of Caspase-3. These target genes were considering related to neuronal apoptosis.4.Detected the expression of the predicted target genes by using Western Blot:Tested on the protein of Bcl-2 and Caspase-3 in hippocampus brain tissue of SE rats and normal rats by using Western-Blot. The results were consistent with the prediction. Bcl-2's expression decreased while the expression of Caspase-3 protein increased. All those would promote neuronal apoptosis.Conclusion.1. Generated the differential expression profiles of miRNAs in SE rat's hippocampus tissue and normal rat's brain tissue.2. Found out that miR-34a, miR-22, miR-125a, miR-21 and miR-29a in the rat brain and peripheral blood have the same changing trends and which are closely related. It provides a basis further research in this field.3. Target genes of MiR-34a and miR-125a would be the Bcl-2 and target genes of miR-21 would be Caspase-3 which participated in the SE regulation of neuronal apoptosis.