The Role Of MicroRNA-134 In Perioperative Ischemic Stroke And Its Molecular Mechanism

Objective: To explore the function and possible molecular mechanisms of microRNA-134(miR-134) in perioperative ischemic stroke.Methods: This study intended to use cellular oxygen-glucose deprivation(OGD) and the mouse middle cerebral artery occlusion(MCAO) models to simulate ischemia. This study was processed by using research techniques such as biochemistry, molecular biology, neuroethology, morphology and so on.1. The miR-134 expression and neuron damage in brain tissue of rat and neurons in culture at different time after ischemia were studied. At the cellular level, we made ischemic cells injury model using 1h OGD. This study was trouped into normal group and 1h OGD/reoxygenation group, the later included four groups: 12 h, 1d, 3d, 7d after reoxygenation. At the animal level, we used ischemic stroke model made by 1h MCAO. The experiment were trouped into normal group, sham group and 1h MCAO/ reperfusion group, the later included four groups: 12 h, 1d, 3d, 7d after reperfusion. RT-PCR was applied to test miR-134 expression at different reoxygenation times after 1h OGD in primarily cultured cerebral cortex neurons, as well as, at different reperfusion times after 1h MCAO in rat brain. We used MTT to test the survival of neurons and Nissl's staining to test neuronal loss of rat brain. Western blotting was used to detect the activation level of Caspase-3, i.e. the expression level of cleaved-caspase-3, to reflect the situation of cell apoptosis.2. The function and mechanism of miR-134 in ischemic brain injury were studied.(1) At the cellular level, ischemic cells injury model was made using method of 1h OGD/24 h reoxygenation, the experiment was grouped into normal group, non-trans group, miR ctrl group, pre-miR-134 group and pre-miR-134 inhibitor group. Firstly, RT-PCR was used to test the miR-134 expression in primarily cultured cerebral cortex neurons after infecting with virus vector. And then MTT was operated to test the ischemic damage of primarily cultured cerebral cortex neurons after 1h OGD/24 h reoxygenation in different infected groups. The influence of miR-134 on apoptosis of primarily cultured cerebral cortex neurons, caused by OGD, was determined by using TUNEL and testing activation level of Caspase-3. Dual Luciferase Reporter Gene Assay was used to test whether the mi R-134 and 3'-UTR sequence of mRNA of its predicted target genes had directly acting site, then selected the target genes. Western blotting was used to detect the regulating roles of miR-134 overexpression or inhibition in HSPA12 B. Finally, cotransfeced HSPA12 B siRNA with pre-miR-134 or miR-134 inhibitor into cells, MTT and Western blot were used to further verify the function of miR-134 in ischemic damage of primary neurons and the regulating effects of miR-134 for HSPA12 B.(2) At the animal level, 1h MCAO/ 24 h reperfusion model was used to simulate ischemic stroke. The experiment was grouped into sham group, MCAO group, MCAO + Pre-miR-134 inhibitor group and its control group. RT-PCR was used to test the miR-134 expression after injecting virus vector in cerebral cortex. The influence of miR-134 on the degree of ischemic reperfusion injury in rat brain was evaluated by neurologic behavior scores, Nissl staining and TTC staining. Western blotting was used to test the influence of miR-134 on expression of its predicted target genes, i.e. HSPA12 B, in rat ischemic brain tissue.Results: 1. After primarily cultured cerebral cortex neurons 1 h OGD, miR-134 expression level obviously increased while cell viability obviously reduced during 12h-7d of reoxygenation when compared with normal group, which was most obviously on 1d. Compared with normal group, miR-134 expression level was highest on 1d of reperfusion in 1h MCAO rat model, and then the expression level gradually reduced; the loss of nerve cells was most serious, and then the loss gradually relieved; the activation level of caspase-3 was highest, and then the activation level gradually reduced. The differences of miR-134 expression and neuron damage in sham group had no statistical significance. 2.(1)Comparing with normal group, miR-134 expression level significantly increased, cell viability obviously reduced, HSPA12 B level and luciferase activity obviously reduced in the pre-miR-134 group when mentioned cerebral ischemia injury caused by OGD. In the pre-miR-134 inhibitor group, miR-134 expression level significantly reduced, while cell viability, HSPA12 B level and luciferase activity obviously increased.But the differences in mRNA of HSPA12 B among these groups had no statistical significance.The differences in luciferase activity of HSPA5 had no statistical significance. After cotransfection of HSPA12 B siRNA, HSPA12 B expression level obviously reduced, cell viability became lower than only transferred pre-miR-134 and schizolytic Caspase-3(activated) in neuron significantly increased.(2) Compared with MCAO group, miR-134 inhibitor group significantly reduced neurobehavioral score, infarct volume, neurons loss and the cl-caspase-3 expression level. However, HSPA12 B protein level was significantly increased compared to MCAO group.Conclusions: miR-134 plays an important role in neuronal ischemic injury, caused by OGD and MCAO, by negatively regulating HSPA12 B expression level. It provides experimental basis, effective biomolecule markers and new therapeutic targets for early prevention and prognosis of perioperative ischemic brain injury. Meanwhile, it further enriches peoples' awareness in the mechanism of cerebral ischemia/hypoxic damage.