| Part One:MiRNA expression profile in epiletic rat hippocampus, qPCR confirmation and bioinformatics on differentially-expressed miRNAsObjectiveBy using microRNA microarray technology, the study was aimed to explore the microRNA expression profile in temporal lobe epilepsy rat hippocampus and screen for the differentially-expressed miRNAs. Then quantitative real-time PCR was used to confirm some of the differentially expressed miRNAs and also to detect the expressional status of the above mentioned miRNAs in rat peripheral blood for comparison analysis with their counterparts in rat hippocampus. After that the study had also used bioinformatics analysis on those differentially expressed miRNAs so as to predict their potential target genes, enriched Gene Ontology (GO) terms and enriched KEGG pathways. The study will lay a foundation for further research on miRNAs'role in injuries caused by temporal lobe epilepsy on the central nervous system.Method1. Establishment of temporal lobe epilepsy rat model:6-8weeks old, healthy and male SD rats were chosen and lithium-pilocarpine was given to these rats in order to trigger status epilepticus (SE), and then these rats were monitored either by videos or EEG so as to confirm the occurrence of spontaneous seizures and the successful establishment of temporal lobe epilepsy (TLE) rats. Then hippocampus and peripheral blood samples of TLE rats were taken for comparison with their counterparts in normal SD rats.2. MiRNA microarray analysis and screen of differentially-expressed miRNAs:miRNAs from the hippocampus of TLE rats were extracted and miRNA microarray analysis was performed to establish expression profile of miRNAs in TLE rat hippocampus and compare with that of normal rats for differentially-expressed miRNAs.3. Confirmation of differentially-expressed miRNAs by quantitative real-time PCR:some of the differentially-expressed miRNAs as detected by miRNA microarray were chosen and confirmed by using quantitative real-time PCR. Meanwhile, their expression status in rat peripheral blood was also examed by quantitative real-time PCR for comparison with that in rat hippocampus.4. Bio-informatics analysis including potential target gene prediction, gene ontology (GO) analysis and enriched KEGG pathway analysis were performed. MiRanda and TargetScan were used for target gene prediction whereas Gene Ontology analysis were performed for enriched GO terms and KEGG analysis were performed for enriched KEGG pathways.Results1. Differentially-expressed miRNAs in TLE rat hippocampus:Using miRNA microarray,9up-regulated and15down-regulated miRNAs were detected in TLE rat hippocampus, respectively. The up-regulated or down-regulated miRNAs were defined as the miRNAs whose fold-change was greater than1.5folds with p-value less than0.05.2. Confirmation of differentially-expressed miRNAs by quantitative real-time PCR:Of those detected differentially-expressed miRNAs, three up-regulated miRNAs (miR-146a, miR-210and miR-27a) and two down-regulated miRNAs (miR-135b and miR-33) were chosen for confirmation by using quantitative real-time PCR. Meanwhile, their expression status in rat peripheral blood was also examed by quantitative real-time PCR for comparison with that in rat hippocampus. Quantitative real-time PCR had confirmed miRNA microarray results of the above-mentioned miRNAs. However, it had not confirmed similar expressional change of miRNAs in rat peripheral blood, since the peripheral blood expressional status of the above-mentioned miRNAs had not been differentially-expressed as their counterparts in rat hippocampus.3. Bio-informatics analysis on the differentially-expressed miRNAs as detected by miRNA microarray analysis:potential target gene prediction was performed using software of TargetScan; enriched GO terms were obtained using GO analysis; enriched KEGG pathways were gained using KEGG pathway analysis.Conclusion1. Temporal lobe epilepsy (TLE) rat model had been established and expression profile of miRNAs in TLE rat hippocampus had been gained.2. There were24differentially-expressed miRNAs in hippocampus of TLE rats as detected by miRNA microarray, among which9up-regulated miRNAs and15down-regulated miRNAs were detected, respectively.3. Some of the differentially-expressed miRNAs were confirmed by quantitative real-time PCR (miR-146a, miR-210, miR-27a, miR-135b and miR-33). However, quantitative real-time PCR had not confirmed similar expressional change of these miRNAs in TLE rat peripheral blood, and a possible linkage between rat hippocampal and peripheral blood miRNAs had not been established.4. Bio-informatics analysis on the differentially-expressed miRNAs as detected by miRNA microarray analysis had obtained the potential target genes, enriched GO terms and KEGG pathways. They were all proposed to be associated with status of temporal lobe epilepsy and the process of epileptogenesis. Part Two:Dynamic changes of rat hippocampal miR-34a and miR-9expression pattern in epileptogenesisObjectiveTo establish temporal lobe epilepsy (TLE) rat model and select the important time points in epileptogenesis before detection of expressional status of an apoptotic-related miR-34a and a neurogenesis-related miR-9by using quantitative real-time PCR. Through comparison of expressional status of miR-34a and miR-9at different time points in TLE or normal rat hippocampus, it was possible to study differential expression of miRNAs under TLE condition, which would lay a foundation for further research on the role of apoptotic-related miR-34a and neurogenesis-related miR-9in epileptic mechanisms.Method1. Establishment of temporal lobe epilepsy rat model:6-8weeks old, healthy and male SD rats were chosen and lithium-pilocarpine was given to these rats in order to trigger status epilepticus (SE), and then these rats were monitored either by videos or EEG so as to confirm the occurrence of spontaneous seizures and the successful establishment of temporal lobe epilepsy (TLE) rats. Time points of1day,7days,14days and2months had been selected for following studies. 2. RNAs had been extracted from TLE rat hippocampus at different time points, and quantitative real-time PCR had been used for detection of the expressional status of miR-34a and miR-9. By comparison of the expression status of miR-34a and miR-9at different time points in TLE rat or normal rat hippocampus, it was possible to study the differential expression of miRNAs at different time points by statistic analysis.Results1. Temporal lobe epilepsy (TLE) rat model had been established and samples of TLE rat hippocampus at different time points had been gained.2. Quantitative real-time PCR and statistic analysis had been used to detect expressional status of miR-34a and miR-9at different time points. The results indicated that the expression of miR-34a had increased at1day,7days,14days and2months (TLE status) post status epilepticus, it had reached a peak at14days post status epilepticus and declined back to the basic level at2months post status epilepticus, although still markedly higher than the normal control group. MiR-9had also exhibited increased an expression level at1day,7days and14days post status epilepticus, it had decreased at2months post status epilepticus (TLE status), reached a peak at14days post status epilepticus and drawn back to a normal level at2months post status epilepticus without statistic significance. ConclusionThe expression change of apoptotic-related microRNA miR-34a and neurogenesis-related microRNA miR-9in epileptogenesis had indicated their involvement in epileptic mechanisms which deserves to be studied and explored in future. |
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