MicroRNA Expression Change In The Hippocampi Of Rats Subjected To Global Ischemia-reperfusion And The Study On The Effect And Mechanism Of Let-7e Upon Caspase-3 Expression

PartⅠMicroRNA expression changes in hippocampus of rats subjected to global ischemia-reperfusionThe expression of many genes changed in the process of global cerebral ischemia-reperfusion, producing a variety of endogenous damage factors, triggering a series of pathological process, resulting in further neuronal injury, which is called "ischemia-reperfusion injury". Hippocampus is very sensitive to ischemia-reperfusion injury, but its mechanism remains unknown.MicroRNA(miR) involved in many biological processes, modulating expression of 1/3 or more animal genes. At present the research on microRNA involving in the regulation of cerebrovascular diseases has just started, and the role of many microRNAs remain unclear. To date, there were no reports regarding the expression profiles of microRNAs in the hippocampus and the changes following global ischemia.ObjectivesTo investigate features of microRNAs expression and changes in hippocampus of rats subjected to global ischemia-reperfusion.Methods1. Global cerebral ischemia-reperfusion rats were produced by the standard four-vessel occlusion (4-VO) method. Adult male Sprague-Dawley rats were divided into four groups:sham,20-min ischemia and 30-min reperfusion(R30min), 20-min ischemia and 24-h reperfusion(R24h), and 20-min ischemia and7-day reperfusion (R7d)2. Brain pathological tests, including HE staining and counting of survival neurons in hippocampal cornu ammonis 1 (CA1), were done in each group.3. Total RNAs of hippocampus in sham, R30min, R24h groups were collected, and their small RNAs were isolated by using centrifugal filter. Then a series steps ofμParafloTM Microarray assay were performed including poly (A) tail plusing, fluorescence labeling, hybridization reaction, hybridization image acquisition and data processing. The rat array chip contained 350 microRNAs, which assembled all mature microRNAs and part of the complementary strand of mature microRNAs (microRNAs*) in the Sanger miRBase Release 11.0.4. To validate the microarray assay results, miR-9, let-7e and miR-29c were selected for intensive analysis by qRT-PCR.5. Several microRNAs were selected to predict their targets by use of bioinformatics database.Results1. Neuronal death following global ischemia-reperfusion was found in a delayedmanner.2. In hippocampus,286 microRNA transcripts were detected, among which 106 had signals>500. Of these, the let-7 family showed highest expression, comprised 32% of total signals, miR-9 and miR-125 also showed high expression.3. In R30min group,266 microRNAs were detected, and 55 revealed statistically significant changes from control. Among these,23 were upregulated and 32 were downregulated. In R24h group,278 microRNAs were detected, and 71 exhibited statistically significant changes. Among these,40 were upregulated and 31 were downregulated.4. Basically, results of real-time RT-PCR were consistent with those of the chip study.5. Based on results of bioinformatics database, it is indicated that caspase-3 might be the target of let-7a, let-7b, let-7c, let-7e, let-7i and let-7g. EIF4E3 and EIF4A1 might be the targets of miR-338. Fos might be the target of miR-7a and miR-7b. EIF4E, EIF5, ATF-1, CREB5 and CREB2F might be the targets of miR-9.ConclusionsThe present study showed that hippocampus had specific microRNAs expression profile, which suggest that microRNA may participate in regulating physiological process within the hippocampus. Many microRNAs changed rapidly and extensively following whole cerebral ischemia-reperfusion injury, suggesting that microRNAs play critical roles in regulating the expression of genes.PartⅡThe study on the effect and mechanism of Let-7 upon Caspase-3 expressionMany evidences suggested that apoptosis is closely related to neuronal injury resulting from anoxia or ischemia. Caspases play a key role in the process of apoptosis, as an important member of the big family of Caspases, Caspase-3 (Casp3) plays an essential role in priming and executing neurocyte apoptosis which leading to cell death. In the first part of the experiment, it was found that let-7e were strongly downregulated at 30min and 24h after reperfusion. Bioinformatics database revealed that Casp3 is the possible predicted target of let-7e. In the next part, we would explore the role of let-7e in protecting against neurocytes apoptosis following anoxia/reoxygenation injury via negatively regulating the expression of Casp3.ObjectivesTo investigate the role and mechanisms of let-7e in protecting against the apoptosis of PC 12 cells in anoxia/reoxygenation (A/R) injury in vitro.Methods1. PC 12 cells were cultured with mouse nerve growth factor(NGF). Precursor let-7e miR (pre-let-7e miR, pre-miR), let-7e miR, nontargeting precursor control (miR-NT), anti-let-7e oligonucleotides (anti-miR, anti-let-7e miR) and nontargeting control inhibitor (anti-miR-NT) were synthesized and transfected into PC 12 cells that were subjected to A/R injury. The experimental groups were devided into control group, A/R group, A/R+NT group, A/R+let-7e miR group, A/R+pre-miR group and A/R+miR+anti-miR group.2. In each group, total RNA was extracted and real-time RT-PCR was performed to detect the mRNA expression level of Cap3, Casp8 and Casp9 and let-7e. Western blotting was performed to analyze the expression level of Casp3, Casp8 and Casp9.3. MTT assay was done to detect the cell survival in each group.4. Flow cytometry was performed to measure cellular apoptosis in each group. Results1. There was a slightly high expression of let-7e in PC 12 induced by NGF and the expression level decreased after A/R injury.2. After A/R injury, the expression of Casp3, Casp8 and Casp9 mRNA and protein increased, together with the percentage of apoptosis increased and the cell viability decreased.3. When subjected to the A/R injury, the transfection of pre-miR or let-7e miR into PC 12 had no effect on the mRNA and protein levels of Casp8 and Casp9, but decreased the mRNA and protein levels of Casp3 and resulted in a decrease of cellular apoptosis as well as the increase of cellular viability. Meanwhile, the transfection of synthetic anti-let-7e abolished these protection effects.ConclusionsLet-7e negatively regulated the levels of Casp3, and thus plays a key role in the protection of PC 12 cell apoptosis under the condition of A/R injury.