| Background:Crouzon syndrome is a syndrome of premature closure of cranial sutures that can lead to severe craniomaxillofacial malformations in children.FGFR2(fibroblast growth factor receptor 2)gene mutation is closely related to the pathogenesis of syndromic craniosynostosis.C278F or C342Y-FGFR2 mutation may lead to Crouzon syndrome.Dura mater plays an important role in the regulation of cranial suture development.However,the underlying molecular biological mechanisms associated with the pathogenesis of Crouzon syndrome have rarely been studied.This study explored and analyzed the effect of over-expression of FGFR2 in dura cells on the biological function of osteoblasts and its potential molecular biological mechanism.Methods:In the first part of this subject,the peripheral blood of the children with Crouzon syndrome and its family members was collected,and the family disease was investigated.Next,the collected blood samples were sequenced by whole exome sequencing(WES),the disease-related mutant genes and "harmful genes" were screened,and the disease-related signal pathways were preliminarily explored.In the second part of this project,we constructed FGFR2 gene mutation transfection complex.Dural cells and skull osteoblasts from 6-day-old C57BL/6 mice were cultured.C278F-and C342Y-FGFR2 gene mutation transfection complex was constructed by lentivirus.After successful transfection of dura cells and gene mutation transfection complex,they were co-cultured with osteoblasts in trans-well system.Two experimental groups and two control groups were set up,namely Oste group,Oste+Dura-vector group,Oste+Dura-C278F group and Oste+Dura-C342Y group.The effect of dura cells transfected with mutant gene on osteoblast cell cycle was detected by flow cytometry.CCK8 and EdU staining experiments were used to evaluate the difference of proliferation level of co-cultured osteoblasts in different groups.The expression of factors related to cell proliferation,differentiation and apoptosis was detected by western blot and RT-qPCR.The third part of this subject focuses on the potential molecular biological mechanism of Crouzon syndrome based on the disease model.Western blot and RT-qPCR were used to detect and analyze the expression levels of selected factors in the Oste group,Oste+Dura-vector group,Oste+Dura-C278 group and Oste+Dura-C342Y group.The selected factors are the key factors in Hippo/YAP-PI3K-AKT signaling pathway.Finally,the rescue experiment was carried out by siRNA.We used siRNA to perform RNA interference experiment(YAP-siRNA)on osteoblasts.In this experiment,osteoblasts without other treatment(Oste),siRNA vector control group(Oste+blank-vector)and YAP-siRNA experimental group(Oste+siRNA)were set,and the expression differences of osteoblast related factor mRNA and/or protein in different groups were detected.In order to further explore the effect of over-expression of FGFR2 in dura cells on the biological function of osteoblasts in co-culture experiments,we perform the rescue experiment of FGFR2-siRNA in dura cells.Dura cells without other treatment(Dura),siRNA control group transfected with empty vector(Dura-control),and dura cells experimental group transfected with FGFR2 siRNA(Dura-siRNA)were set in this experiment.The proliferation level of osteoblasts in different groups was detected by CCK8.The mRNA and/or protein expression of osteogenesis-related factors in different groups were detected by western blot and RT-qPCR.In order to explore whether proteasome is involved in YAP degradation,osteoblasts without other treatment(Oste),non-targeted control siRNA of siRNA empty vector(Oste+blank-vector),experimental group with YAP-siRNA(Oste+siRNA)and experimental group with YAP-siRNA and MG132(Oste+siRNA+MG132)were set in this part of the experiment.Results:In the first part,we collected the peripheral blood of the patients with Crouzon syndrome,twenty-eight high-quality and reliable gene variants were found in the studied families.We labeled them as pathogenic gene mutations(twenty-three missense mutations,one unknown mutation,three non acquired mutations and one synonymous mutation).According to phenolyzer analysis and literature review,only FGFR2 gene mutation is closely related to the pathogenesis of craniosynostosis.In order to further verify the accuracy of whole exome sequencing results,Sanger sequencing was used for verification.In addition,through the enrichment analysis of KEGG pathway,multiple signal pathways including PI3K-AKT may play an important role in the pathogenesis of the disease.In the second part of the experiment,we first identified and cultured dural cells and osteoblasts,then successfully constructed dura cells with C278F-FGFR2 point mutation and C342Y-FGFR2 point mutation,and successfully co-cultured with osteoblasts in the trans-well system.The results of flow cytometry showed that the proportion of osteoblasts in S phase and G2/M phase in Oste+Dura-C278F group and Oste+Dura C342Y group was significantly higher than that in control groups.The results of CCK8 experiment showed that the proliferation level of osteoblasts in Oste+Dura-C278F group and Oste+Dura-C342Y group was significantly higher than that in the control groups,and showed a time-dependent increasing trend.EdU staining showed that the proliferation level of osteoblasts in Oste+Dura-C278F group and Oste+Dura-C342Y group was also significantly higher than that in control groups.The results of RT-qPCR and western blot showed that the expression levels of proliferation related factors(PCNA),cell cycle related factors(CDK1,CDK2,CDK4)and differentiation related factors(ALP,RUNX2,Osteopontin,Osteocalcin)in osteoblasts(Oste+Dura-C278F and Oste+Dura-C342Y groups)were significantly up-regulated,while the expression levels of apoptosis related factors(BAX,BCL2,Caspase3)were not significantly different from those in control groups.In the third part of the experiments,the results of RT-qPCR and western blot showed that the expression levels of YAP,p-PI3K,p-AKT1 mRNA and/or protein in Oste+Dura-C278F and Oste+Dura-C342Y groups were significantly increased,and the expression levels of LATS2 and p-YAP were significantly decreased.However,the mRNA and/or protein expression levels of PI3K and AKT1 were not significantly different in all groups.In the experiment of YAP-siRNA,the results of RT-qPCR showed that the expression level of YAP mRNA in osteoblasts was significantly decreased,but there was no significant difference in the expression levels of PI3K and AKT mRNA in the three groups.The results of western blot showed that the expression levels of p-PI3K and p-AKT1 in experimental groups were significantly decreased.In the experiments of FGFR2-siRNA,the results of CCK8 experiment showed that the proliferation of osteoblasts composed of Oste+Dura-siRNA was significantly inhibited.The results of RT-qPCR and western blot showed that in Oste+Dura-siRNA group,the mRNA and/or protein expression levels of FGFR2,PCNA,YAP,TEADsl-4,p-PI3K and p-AKT1 decreased significantly,while the expression levels of LATS2 and p-YAP increased significantly.In the experimental part of exploring proteasome with MG 132,the results of western blot showed that the down-regulated level of YAP expression was significantly alleviated after osteoblasts co-cultured with dura cells transfected with FGFR2-C278F and FGFR2-C342Y were treated with MG 132.Conclusions:1.The whole exon sequencing results confirmed that FGFR2 gene mutation was closely related to the pathogenesis of Crouzon syndrome.At the same time,it was found that multiple signal pathways including PI3K-AKT may play an important role in the pathogenesis of the disease.2.FGFR2 Crouzon mutation(C278F-FGFR2 and C342Y-FGFR2)in dura cells can improve the proliferation and differentiation of co-cultured osteoblasts.The dura cells transfected with C278F-FGFR2 and C342Y-FGFR2 point mutations can accelerate the cell division of osteoblasts by affecting the cell cycle activity of co-cultured osteoblasts,and then promote the proliferation of osteoblasts.3.Crouzon mutant dura cells may regulate osteoblast proliferation by regulating Hippo/YAP and PI3K-AKT signaling pathway,and further affect the disease process of Crouzon syndrome.. |
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