The Effects And Molecular Mechanism Of Lentivirus-mediated Integrin β1Silencing In Sombatic Cell Culture Model Of Refractory Epilepsy

PART1GENE EXPRESSION STUDIES OF INTEGRINβ1IN SOMBATIC RAT CELL CULTURE MODEL OFREFRACTORY EPILEPSY BY REAL-TIME QPCRObjective To investigate the expression of Integrin β1, and further explorethe role in the Sombati cell culture model of refractory epilepsy.Methods The hippocampal neurons were isolated from1-day rat andcultured in Serum-free B27/neurobasal medium. On the14th day, the neuronswere divided into control and experimental groups randomly. According toSombati’s procedures, the neurons were cultured in low Mg2+pBRS for threehour to mimic status epilepticus injury in experimental groups, and in thecontrol groups, the neurons were cultured in1mM Mg2+pBRS. After then,neurons of each group were cultured in maintenance medium for one day. At theend of the treatment, the mRNA expression level of integrin β1of neurons were detected using Real-time qPCR.Results Compared with the control group, Sombati epileptic cell model inmaintenance medium for four day, integrin β1mRNA expression level increasedsignificantly.Conclusions The expression of integrin β1increase in the Sombati cellculture model of epilepsy, integrin β1may participate in the network ofepileptogenic reorganization together. PART2CONSTRUCTION OF RECOMBINANTLENTIVIRAL VECTOR CONTAINING INTEGRIN β1SHRNAAND ITS EFFECTS ON INTEGRINβ1GENE EXPRESSION INTHE SOMBATI CELL CULTURE MODEL OF REFRACTORYEPILEPSYObjective To construct recombinant lentiviral vector containing integrin β1shRNA and provide an effective tool to study integrin β1gene effect andpossible mechanism of the Sombati cell culture model of refractory epilepsy.Methods (1) According to integrin β1gene sequence, four RNAinterference target sequences of integrin β1were designed using siRNA designsoftware. Four lentiviral plasmid were constructed and sequenced to validate thepositive clones. HEK293T cells were routinely cultured and the cells wereco-transfected with ShRNA lentiviral vector and secondary packaging originalvector plasmid. After measuring virus titer, the virus were concentrated andcollected. PC12cells were routinely cultured and the integrin β1shRNA1-4were transfected into PC12cells. The groups were obtained: integrin β1RNANC group, integrin β1shRNA1group, integrin β1shRNA2group, integrin β1shRNA3group and integrin shRNA4group. The efficiency of PC12cellsinfected with lentivirus was estimated using fluorescence microscope. The effectof integrin β1shRNA silencing in PC12target cells was determined usingWestern blotting assay, in order to filter out the best one of integrin β1shRNAlentiviral vector. Hppocampal neurons were routinely cultured to11th day andwere transfected with Integrin β1shRNA-NC or the best one of silence (Integrinβ1shRNA2lentivirus). And then the neurons were continually cultured to14th day. According to Sombati’s procedures, the neurons transfected with integrin β1shRNA-NC were cultured in low Mg2+pBRS or1mM Mg2+pBRS for three hour.At the same time, the neurons transfected with integrin β1shRNA2lentiviralvector were cultured in low Mg2+pBRS or1mM Mg2+pBRS for three hour. Thegroups were obtained: normal hippocampal neurons+integrin β1-NC, normalhippocampal neurons+integrin β1shRNA2, Sombati epileptic cellmodel+integrin β1-NC, Sombati epileptic cell model+integrin β1shRNA2. Afterthen, neurons of each group were cultured in maintenance medium. At the endof the treatment, the effect of integrin β1gene silencing in normal hippocampalneurons or Sombati epileptic cell model was detected using Western blottingassay.Results RNAi lentivirus expression vectors targeting rat integrin β1genewere successfully constructed and confirmed by DNA sequencing. Therecombinant lentivirus particles were packaged successfully to produce enoughtiter for the following experiments. After the neurons transfected with integrinβ1shRNA lentiviral vector, the expression of integrin β1significantly decreasedin the rat hippocampal neurons and rat Sombati epileptic cell model.Conclusion The recombinant lentiviral vector containing integrin β1shRNA was constructed successfully and the gene-silencing effects wereeffective and stable in neonatal rat hippocampal neurons and Sombati epilepticcell model. PART3STUDY ON PROTEIN EXPRESSION OFFAK-MEDIATED SIGNALING PATHWAYS IN SOMBATICELL CULTURE MODEL OF REFRACTORY EPILEPSYAFTER INTEGRIN β1GENE SILENCINGObjective Sombati epileptic cell model were transfected with integrin β1lentiviral vector to explore the role and mechanism of integrin β1signalingpathways in epilepsy in order to provide scientific basis for the exactpathogenesis of epilepsy.Methods The hippocampal neurons were isolated from1-day rat andcultured in Serum-free B27/neurobasal medium. The neurons were divided intodifferent groups randomly: normal hippocampal neurons (control), Sombatiepileptic cell model, Sombati epileptic cell model+integrin β1shRNA-NC,Sombati epileptic cell model+integrin β1shRNA2. Hppocampal neurons of allgroups were routinely cultured to11th day and were transfected with integrin β1shRNA-NC in Sombati epileptic cell model+integrin β1shRNA-NC group andintegrin β1shRNA2in Sombati epileptic cell model+integrin β1shRNA2group.And then the neurons were continually cultured to14th day. According toSombati’s procedures, the neurons of Sombati epileptic cell model, Sombatiepileptic cell model+integrin β1shRNA-NC, Sombati epileptic cellmodel+integrin β1shRNA2were cultured in low Mg2+pBRS for three hour. Atthe same time, the group of normal hippocampal neurons (control) was culturedin1mM Mg2+pBRS for three hour. After then, neurons of each group werecultured in maintenance medium for four days. At the end of the treatment, theassociated proteins involved in signaling pathways such as Phospho-FAK (Tyr397), Phospho-Rac1/cdc42(Ser71), Phospho-PAK1(Ser199/204)/PAK3wasdetected using Western blotting assay.Results Compared with normal hippocampal neurons, the expression levelof Phospho-FAK was significantly higher in Sombati epileptic cell model.Compared with Sombati epileptic cell model, the expression level ofPhospho-FAK was significantly lower in the neurons of Sombati epileptic celltransfected with integrin β1shRNA2. The expression level ofPhospho-Rac1/cdc42(Ser71), Phospho-PAK1(Ser199/204)/PAK3had nosignificant changes in normal hippocampal neurons, Sombati epileptic cellmodel, Sombati epileptic cell model transfected with integrin β1shRNA-NC orintegrin β1shRNA2.Conclusion Integrin β1plays a role in Sombati cell culture model depends onthe activation of FAK-related signaling pathways, which activates Tyr397phosphorylation sites, not including the activation ofPhospho-Rac1/cdc42(Ser71) and Phospho-PAK1(Ser199/204)/PAK3.