| Focal cortical dysplasia(FCD) is a pathological type of cortical dysplasia(MCD),which is an important cause of refractory epilepsy.According to statistics,80% of children with refractory epilepsy under 3 years have MCD.More than 75% of children treated surgery were pathologically confirmed to have FCD.In 1971,Taylor et al observed surgical resection specimens of 10 refractory epilepsy patients and found abnormal cortical structures with the pathology of dysmorphic neurons and balloon cells,which first proposed the concept of "focal cortical dysplasia" and described the pathological tissue structural features of FCD.This concept has been widely used in Anatomy,Pathology,Neuroimaging and other majors.With the continuous understanding of FCD,the pathological classification standard of FCD is constantly updated,especially in 2004,the classification standard proposed by Palmmini et al has been widely used in clinical,in which the FCD was classified as type I or II.However the reproducibility of the classification method has been highly controversial.In 2011,the International League Against Epilepsy(ILAE)reclassified the FCD,modified the Palmini classification,and added the concept of the FCDⅢ type.There are four subtypes of FCD,FCDⅢa(FCD with hippocampal sclerosis),FCDⅢb(FCD with neurological tumors),FCDⅢc(FCD with vascular malformation)and FCDⅢd(FCD with brain injury,inflammation,perinatal hypoxia and other early brain lesions).The pathogenesis of FCD may be closely related to the proliferation,migration and differentiation of neurons in early embryonic development.The human cerebral cortex forms in the 8 to 24 weeks of embryonic development.During the cortex development,excitatory neurons migrate from the ventricle to the cortical area.Meanwhile,inhibitory neurons migrate tangentially from the ganglion processes to the cortex.When neurons reach a particular location,they start to send out axons and dendrites,thus forming a neural network.Any adverse factors,such as infection,hypoxia,trauma,gene mutation,can affect the normal migration trajectory of neurons and the formation of a normal neural network.In the past decades,with the popularization of Neuroimaging,Genetics and Genetic sequencing technologies,more and more studies have focused on the pathogenesis and epilepsy mechanism of various subtypes of FCD.Scholars have proposed many hypotheses about the pathogenesis of FCD,including transmitter receptor hypothesis,GABA receptor hypothesis,GABA synaptic activity pacemaker hypothesis,mTOR pathway hyperactivation hypothesis,somatic mutation hypothesis and virus infection,etc.Clarify the pathogenesis of FCD and related epileptogenesis will be assistant to improve the diagnosis and personalized treatment of intractable epilepsy.The key role of the mammalian target of rapamycin(mTOR)signaling pathway in refractory epilepsy is gradually being recognized,especially after its inhibitors were approved for the treatment of Tuberous Sclerosis.Due to the high similarity between TSC and FCD in terms of histopathology and clinical manifestations,mTOR signaling is also increasingly studied in FCD.Signal transducers and activators of transcription 3(STAT3)is a DNA-binding protein,with transcriptional ability,which can participate in cell proliferation,differentiation,apoptosis and other biological phenomena in vivo biological signal through different signaling pathways.STAT3 is also an important downstream molecule of the mTOR pathway.The mTOR-STAT3 signaling pathway can mediate gliosis,apoptosis,stem cell proliferation and differentiation,and autophagy.In recent years,it has been found that STAT3 is closely related to the etiopathogenesis of epilepsy,especially to apoptosis in hippocampal neurons.This study intends to detect the expression of the mTOR-STAT3 signaling pathway to explore the pathogenesis of FCD.For FCD typeⅡI,because FCDⅡIa can clearly distinguish hardened hippocampus with surrounding FCD during surgery,the FCDⅢb,FCDⅢc,FCDⅢd can not distinguish from the primary lesion tumor,vascular disease or early brain changes.So the study choose FCDⅡIa patients as the objects to explore the pathogenesis of FCD typeⅡI.With the development of precision medicine,searching for the candidate pathogenic genes of FCD from the perspective of genetics has become the hotpot of the current clinical research.There has found more than 100 kinds of gene mutations related to MCD,but due to MCD disease usually complex,there are rare results about FCD candidate gene.Currently,TSC alone has clear genetic mutations,precise mutated genes have not yet been found for other types of MCD.Therefore,the deep exploration of FCD can help to study the pathogenesis of FCD and epileptogenesis.FCD typeⅡ is a common pathological type in therapeutic resection brain tissue in patients with refractory epilepsy.It was mostly found in the areas outside of temporal lobes,especially in the frontal lobe.On Imaging,it generally shows cortical thickening,abnormal development of sulcus and gyrus,and unclear boundaries of gray and white matter.Although about 60% of the children with FCDⅡ have good control after surgery,40% of the patients still have recurrent seizures postoperative.A growing number of studies have found suspicious genetic mutations in FCD typeⅡ,however,its molecular genetic etiology remains unknown.However,the molecular genetic etiology is still unclear.With the wide popularization of precision medicine,it became possible to find the FCD related pathogenic genes,and this study intends to use the method of biological information analysis to find the potential pathogenic genes of FCDⅡ.Part One Bioinformatics Analysis of FCD typeⅡ gene expressionObjective: To identify the potential pathogenic genes of FCDⅡ by using the method of bioinformation analysis,and to reveal the molecular mechanism of FCDⅡ at the molecular level.Methods: We downloaded two datasets for FCDⅡ from the Gene Expression Omnibus data repository.Differentially expressed genes(DEGs)between FCDⅡ and normal brain tissues were identified,and functional enrichment analysis was performed.A protein–protein interaction network was constructed,and hub genes were identified from the DEGs.The hub gene expression was validated using Western blot in vitro.IHC staining was performed to verify the feasibility of the target molecular markers identified in the bioinformatics analysis.The FCDⅡ mRNA expression profile datasets GSE128300(GPL10999platform)and GSE62019(GPL10558 platform)were downloaded from the National Center for Biological Data Center GEO database.Differentially expressed genes between FCD typeⅡ and normal brain tissues were identified using the Limma package in R language,and GO and KEGG functional enrichment analysis were performed using Metascape software and David tool.STRING database(https://string-db.org)was used for building protein-protein interaction network PPI,and hub genes were identified from DEGs using the Cytoscape program.Then,12 surgical brain tissue samples of FCDⅡ children were selected as experimental groups,and 5 children with autopsy and neurosurgery intracranial decompression were selected as control groups to verify the hub genes screened in vitro.The expression of the selected hub genes in both groups was verified by RT-PCR,Western blot and immunohistochemistry.Results:1.One hundred sixty-seven common DEGs were identified between the datasets downloaded from the GEO database;2.The GO and KEGG analyses showed that variations were prominently enriched in some functions associated with gene expression,such as Fanconi anemia pathway,Hippo signaling pathway,myelin assembly and regulatory of the cytoskeleton.Fanconi anemia pathway enrichment is the most obvious;3.Five hub genes(FANCI,FANCA,BRCA2,RAD18 and KEAP1)were identified by Cytoscape;4.The expression of the five hub genes in FCDⅡ and control brain tissue samples tested by RT-PCR,the results showed that the five hub genes(FANCI,FANCA,BRCA2,RAD18 and KEAP1)were significantly more expressed in FCDⅡ compared with normal brain tissues(P<0.05);5.Western blot confirmed that all hub gene expressions were higher in the FCDⅡ tissue than in the normal brain tissue(P<0.05);6.IHC staining showed that the FANCI expression significantly increased in the FCDⅡ tissue.Conclusion:1.There are DEGs between FCD typeⅡ and normal brain tissues,which are mainly concentrated in the Fanconi anemia pathway,myelination process,cytoskeleton regulation process,Hippo signaling pathway and other biological processes.2.There are DEGs between FCDⅡ and normal brain tissues,which may be considered biomarkers for FCDⅡ.3.The DEGs and hub genes identified in the bioinformatics analysis could serve as candidate targets for diagnosing and treating FCDⅡ.Part Two Expression of mTOR-STAT3 signaling in FCD subtypesObjective:To investigate the expression of mTOR-STAT3 signal pathway in different subtypes of FCD and explore the pathogenesis of FCD.Methods: 38 children with FCD who underwent surgical treatment were retrospectively reviewed from 70 children with FCD related refractory epilepsy.11 children with FCDI,12 children with FCDⅡ and 15 children with FCDⅢa.The study chose 5 children who underwent intracranial decompression as control group.HE staining was applied to detect the histomorphology of the experimental and control groups.The expression of mTOR,P-mTOR,P-70s6 k,STAT3,P-STAT3 were detected in brain specimens by western blot and immunohistochemistry.Results:1.HE staining results showed the morphological characteristics of FCDI,FCDⅡ and FCDⅢa brain tissues,which were met the diagnostic criteria of ILAE in 2011;2.P-mTOR,P70s6 k and P-STAT3 expressed higher in FCDⅡ and FCDⅡIa groups,which expressed lower in FCDI type and control group;3.There is a statistical difference on the expression of P-mTOR,P70s6 k and P-STAT3 between FCDI and FCDⅢa groups;4.The expression of P-STAT3 and P-mTOR in hippocampus of FCDⅢa was significantly higher than in FCDⅢa cortex;5.There was a significant and positive correlation between P-mTOR and P-STAT3 expression in FCD typeⅡ and FCDⅢa;6.The expression of P-STAT3 and P-mTOR was significantly higher in the FCDⅢa hippocampus than in the FCDⅢa cortex.Conclusions:1.P-mTOR,P70s6 k and P-STAT3 were upregulated in FCD typeⅡ and FCDⅢa brain tissues,and the mTOR-STAT3 signaling pathway was hyperactivated.2.The mTOR-STAT3 signaling pathway has different activity in FCDI and FCDⅢa,FCDI and FCDⅢa may have different pathogenesis.3.Hippocampal sclerosis in FCDⅢ a may be the key to the FCDⅢa.Full text conclusions:1.There are DEGs between FCD typeⅡ and normal brain tissues,which are mainly concentrated in the Fanconi anemia pathway,myelina tion process,cytoskeleton regulation process,Hippo signaling pathway a nd other biological processes.2.There are DEGs between FCDⅡ and normal brain tissues,whic h may be considered biomarkers for FCDⅡ.3.The DEGs and hub genes identified in the bioinformatics analysi s could serve as candidate targets for diagnosing and treating FCDⅡ.4.P-mTOR,P70s6 k and P-STAT3 were upregulated in FCD type I I and type FCDⅢa brain tissues,and the mTOR-STAT3 signaling path way was hyperactivated.5.The mTOR-STAT3 signaling pathway has different activity in F CDI and FCDⅢa,FCDI and FCDⅢa may have different pathogenesis.6.Hippocampal sclerosis in FCDⅢ a may be the key to the FCDⅢ a. |
