Proteomics Analysis Of Plasma And Functional Identification Of Gene From Patients With Epilepsy

Epilepsy is a common chronic disease in clinical.The age of onset is variable.Epilepsy is characterized by recurrent and various forms of seizures,which has different effects on the body,spirit and intelligence of patients.Epilepsy can be manifested as a variety of epilepsy syndrome,and can also evolve into the status of epilepsy,even cause sudden epileptic death.The pathogenesis of epilepsy is complex and has not yet been clarified.Cell dysfunction caused by related gene mutation may be one of the causes.In addition,in recent years,omics technology has been more and more widely used in the field of medicine,and the rapid development of proteomics,as an important part of omics technology,has provided powerful technical support for the identification of clinical biomarkers,early diagnosis of diseases,and targeted intervention.However,at present,most of the proteomics studies on epilepsy focus on animal models or human brain tissue samples,but few on plasma proteomics.In this study,on the one hand,quantitative proteomics combined with bioinformatics technology was used to identify plasma differentially expressed proteins in epileptic patients;on the other hand,vitro cell experiments were conducted to identify the mechanism of cell apoptosis induced by the elongate mutation of pathogenic gene CACNA1 A.The specific work is as follows:⒈TMT-based proteomic analysis of plasma from children with Rolandic epilepsy Objective: TMT-based proteomics technology was used to analyze the differentially expressed proteins in the plasma of children with Rolandic epilepsy,and the differentially expressed proteins were screened and visualized using bioinformatics technology to search for possible biomarkers in the plasma.Methords: Plasma was collected from children with Rolandic epilepsy（case group: 5 cases）and children with migraine（control group: 5 cases）.Then,protein extraction and enzymatic digestion were performed.The peptides after enzymatic hydrolysis were labeled with TMT reagent,then graded by high performance liquid chromatography（HPLC）,and analyzed by liquid chromatography-mass spectrometry（LC-MS/MS）.Maxquant（v1.5.2.8）was used for retrieval,and the database was Swiss Prot Human for differentially expressed proteins identification.On the basis of qualified quality control,differentially expressed proteins with fold change >1.2 or fold change <0.83,P value < 0.05,and up-regulated/down-regulated expression in two or more groups were selected as DEP for bioinformatics analysis: Including GO analysis,KEGG pathway analysis,establishment of protein-protein interaction（PPI）network,and hub gene screening of DEP.Results: A total of 752 proteins（670 quantitative proteins）were identified.Among them,217 differentially expressed proteins existed in the samples of two or more groups,in which the up-regulated DEP were 46 and down-regulated DEP were 111.These proteins are associated with immune or inflammatory responses,activation of complement cascades,lipid and glycolysis metabolism,abnormal regulation of fibrinogen and fibrinolytic systems,and oxidative stress.A total of 20 hub DEP were identified,among which fibronectin,alpha-1 acidic glycoprotein family（AGP1,AGP2）,serum amyloid P,fibrinogen chain（γ and α）,complement C9,complement C8,complement C4 B,complement factor I were up-regulated,while Glyceraldehyde 3-phosphate dehydrogenase（GAPDH）,triose phosphate isomerase（TIM）,glyceric acid phosphate kinase 1（PGK1）,fructose diphosphate aldolase（ALDOA）,protein disulfide isomerase（PDI）,catalase（CAT）,superoxide dismutase [Cu-Zn]（h Sod1）,heat shock protein 90 beta 1,Alpha-enolase,apolipoprotein A1 were down-regulated.The upregulated expression of alpha-1 acidic glycoprotein（AGP）and serum amyloid P（SAP）in the plasma of children with Rolandic epilepsy was reported for the first time.⒉Mechanism of apoptosis induced by CAG elongate mutation of PME pathogenic gene CACNA1 A in SH-SY5 Y cellsObjective: We found two CACNA1 A gene homozygous mutations（47 exon c.6975₆976ins CAG）in a third-generation family with progressive myoclonic epilepsy（PME）accompanied by progressive ataxia,hypotonia and cognitive decline.This study is to investigate the effect of the 3 ’-terminal CAG elongate mutation of PME pathogenic gene CACNA1 A on the apoptosis of SH-SY5 Y cells to provide a basis for research of the pathogenesis of PME.Methords: DNA molecules expressing Cav2.1wt（Q13）,Cav2.1mt（Q26）,Cav2.1N,Cav2.1dm1,Cav2.1dm2,Cav2.1C,Cav2.1dm3,Cav2.1dm4 and Cav2.1dm5 were constructed,and each corresponding DNA molecule was cloned into a pc DNA3.1 neo vector with restriction endonuclease sites of Bam H I and Xba I.Lipofectamine 3000 was used to transfect SHSY5 Y cells.After 48 hours,wst-1 cell proliferation kit was used to detect cell proliferation.Flow cytometry was used to detect cell cycle and apoptosis.Cell apoptosis morphology was also observed.Cav2.1dm3 G fusion gene was constructed by GFP labeled Cav2.1dm3 DNA molecule.Western blot was used to detect the protein expression of Cav2.1wt,Cav2.1mt and Cav2.1dm3 G in the transfected cells.Immunofluorescence and DAPI staining were used to observe the distribution of protein expression of Cav2.1wt and Cav2.1dm3 G in cells.Western blot was used to detect the protein expression of bcl-2,Bax,cleaved caspase 3 and cleaved PARP in each group of Cav2.1wt,Cav2.1mt and Cav2.1dm3 respectively.Results: Compared with Cav2.1wt（Q13）and control group,Cav2.1mt（Q26）group significantly reduced the cell proliferation（**P<0.01）.Cav2.1N and Cav2.1C group had no significant effect on cell proliferation compared with control group.Cav2.1dm3 group significantly reduced the cell proliferation compared with Cav2.1dm1 and Cav2.1dm2 group respectively（**P<0.01）.Cav2.1dm4 and Cav2.1dm5 group had no significant effect on cell proliferation compared with control group.Cav2.1dm3 group inhibited cell proliferation significantly compared with control,Cav2.1dm4 and Cav2.1dm5 group respectively（**P<0.01）.Cav2.1mt and Cav2.1dm3 group had no significant effect on cell cycle distribution compared with control and Cav2.1wt group.Cav2.1dm3 group significantly increase the percentage of apoptosis compared with Cav2.1wt and control group（**P<0.01）.Cav2.1dm3 group significantly increase the percentage of apoptosis compared with cav2.1mt group（**P<0.01）.Western blot showed that Cav2.1wt protein was mainly expressed in cell membrane（87.0%,**P<0.01）,Cav2.1mt protein was expressed in cell membrane and nucleus（46.7%vs53.3%,**P<0.01）,while Cav2.1dm3 G protein was mainly expressed in nucleus（85.9%,**P<0.01）.The results were consistent with the results of cell immunofluorescence staining.Cav2.1mt and Cav2.1dm3 group showed significantly decreased expression of bcl-2 /Bax ratio,while increased expression of cleaved caspase 3 and cleaved PARP compared with Cav2.1wt group（**P<0.01）.Cav2.1dm3 group showed significantly decreased expression of bcl-2 /Bax ratio,while increased expression of cleaved caspase 3 and cleaved PARP compared with Cav2.1mt group（**P<0.01）.Conclusions:⑴Most of the proteins identified from plasma of children with Rolandic epilepsy by TMT-based quantitative proteomics were related to immune or inflammatory response,activation of complement cascade,lipid and glycolysis metabolism,abnormal regulation of fibrinogen and fibrinolytic system,and oxidative stress response.⑵A total of 20 hub DEP were identified by bioinformatics,among which DEP related to immune or inflammatory response,complement cascade activation,and fibrinogen activation were up-regulated,while DEP associated with lipid and glycolysis metabolism,abnormal fibrinolytic system regulation,and oxidative stress response were down-regulated.The up-regulated expression of alpha-1 acidic glycoprotein（AGP）and serum amyloid P（SAP）in the plasma of children with Rolandic epilepsy was first reported.⑶The mutant Cav2.1 protein encoded by the CAG elongate mutation of pathogenic gene CACNA1 A in epilepsy that contains the extended poly Q sequence in C-terminal may undergo enzymatic hydrolysis due to its unstable structure and produce truncated mutant proteins.The truncated mutant proteins containing the C-terminal were more prone to translocate to nuclear and could induce SH-SY5 Y cell apoptosis through Bcl-2 /Bax/caspase-3/PARP pathway.