Effects And Mechanism Of SiRNA Targeting Notch1on Neuron-like PC12Cells Subjected To Ischemic Injury

Ischemic cerebrovascular disease is the most common clinical neurologicaldisease, and ischemic cerebral injury is its important pathological and pathophysiolo-gical processes. In recent years, studies on the mechanism of cell signal transductionpathways involving in ischemic brain damage and repair have received extensiveattentions, and more and more signal pathways were found to be involved in theprocess of cerebral ischemia injury.The Notch1signal is a highly conserved signal transduction pathway in theevolution process. Research has shown that Notch1signal can regulate the generationof arteries and involve in the neonatal process of neurons after ischemic cerebralinjury[1], but it is still not clear as to the changes of Notch1signaling pathway and itsmechanism in the process of cerebral ischemic injury. This study established themodel of cerebral ischemia injury in vitro, explored the early effects and mechanismof Notch1signal pathway in cerebral ischemic injury, which lays a foundation for theresearch on the mechanism of Notch1signaling pathway in cerebral ischemic injury.Furthermore, the research revealed the relationship and internal mechanism betweenNotch1signal transduction pathway and cerebral ischemic injury, which may providetheoretical basis and therapeutic target for the therapy of cerebral ischemic injury.1.Model of neuron-like PC12cells subjected to oxygen-glucose deprivation invitroObjective: To induce and cultivate neuron-like PC12cells by NGF（150ng/ml）stimulation, perform oxygen and glucose deprivation （OGD）, and build cerebralischemic injury model in vitro.Methods: Sugar-free culture medium and hypoxia box were used to OGD; theOGD model was evaluated by MTT and immunofluorescence staining method.Results: PC12cells were cultured6d by NGF with the final concentration of150 ng/ml, which manifested a typical neuronal morphology. MAP2immunofluorescencestaining showed strong positive. After OGD neurons manifested injury, necrosis andapoptosis in different degree. With the extension of OGD, the survival rate decreased,and apoptosis rate increased.Conclusions:（1） Neuron-like PC12cells induced by NGF has the properties ofneurons, and can be applied to establish a model of neuron ischemia injury;（2）Successfully established the neuron-like PC12cells model of oxygen glucosedeprivation（OGD） model, the OGD model can be stable analogy ischemic neuronaldamage in vivo, and has high repeatability.2. Notch1-siRNA silenced Notch1mRNA and detected silence effectsObjective: To silence targeting Notch1to neuron-like PC12cells, detect silenceeffects, and select the most efficient interfere sequence, the best transfectionconcentration and the best transfection time.Methods: We took the Notch1gene as a target, designed and synthesized threesiRNA sequences, transfected cells by lipofecamine^TM2000, and detected the effectsof different silence sequences, different concentration and time on the expression ofNotch1mRNA and protein by Realtime-PCR and Western blot technology.Results:（1） After Notch1-siRNA were transfected into neuron-like PC12cellsby lipofecamine^TM2000, the expression of Notch1mRNA and protein weresignificantly decreased than those in the control group （P<0.05）;（2） The expression ofNotch1mRNA and protein decreased significantly （sequence/time/concentration） inNotch1-siRNA2group, transfection concentration2μg/ml group, transfection time48h group compared with other groups（P<0.05）.Conclusion: Lipofecamine^TM2000can successfully transfect Notch1-siRNA intoneuron-like PC12cells, and the Notch1gene was most effectively silenced throughNotch1-siRNA2interference sequence and2μg/ml concentration and48h transfectiontime.3. The expression changes of Notch1signaling pathway in neuron-like PC12cellssubjected to OGD damage and the impact of Notch1-siRNAObjective: To detect the expression changes of Notch1signaling pathway inneuron-like PC12cells subjected to OGD and Notch1-siRNA injury, and explore themode and mechanism of Notch1signal transduction pathway in cerebral ischemic injury.Methods: On the basis of neuron-like PC12cells subjected to OGD12h model invitro, We treated it with Notch1siRNA, detected the expression of Notch1mRNA byRealtime-PCR technology, and detected the expression of Notch1and Notch1intracellular domain （NICD） protein by Western-blot technology.Results:（1） The expression of Notch1mRNA was significantly increased inOGD group when compared with the control group, and was dramatically decreasedin OGD+Notch1-siRNA group when compared with OGD group（P<0.05）;（2） Theexpression of Notch1and NICD protein was significantly increased in OGD groupwhen compared with the control group, and was dramatically decreased inOGD+Notch1-siRNA group when compared with OGD group（P<0.05）.Conclusion: Cerebral ischemic injury can induce the activation of endogenousNotch1signaling pathway, and the high expression of Notch1induced by cerebralischemic injury may be initiating factors for the activation of Notch1signalingpathway.4. Effects and mechanism of Notch1-siRNA on neuron-like PC12cells subjectedto ischemic injuryObjective: To detect the effects of Notch1-siRNA on ischemia-induced nervecells apoptosis and apoptosis-related gene IAP-1and Caspase-3expression, and toexplore endogenous effects and possible mechanism of Notch1signal pathwayliveness induced by the nerve cells subjected to ischemic injury.Methods: FCM was used to identify apoptosis rate. Realtime-PCR and Westernblot were used to detect the expression changes of the apoptosis related genes IAP-1and Caspase-3.Results:（1）The apoptosis rate was significantly increased in Notch1-siRNA+OGDgroup when compared with OGD group （P<0.05）;（2） The expression of IAP-1mRNAand protein was significantly decreased and Caspase-3was significantly increased inOGD group when compared with the control group（P<0.05）;（3） The expression ofIAP-1mRNA and protein was significantly decreased and Caspase-3was significantlyincreased in OGD+Notch1-siRNA group when compared with OGD group（P<0.05）.Conclusions: The activation of endogenous Notch1signaling pathway induced byischemia may be exert anti-apoptotic neuron-protective effects in the specific process of the cerebral ischemia through the up-regulation of IAP-1and inhibition ofCaspase-3expression.