Study On The Mechanism Action Of Spinal Ependymoma Based On TGF-beta Signaling Pathway BMP4/FBN1

Objective: To explore the prognostic factors related to spinal ependymoma patients,explore the differential expression signal pathway in spinal ependymoma,establish the prognosis prediction model of genes related to differential expression signal pathway in ependymoma,and investigate the biological functions and regulatory mechanisms of target genes and their proteins in the differential expression signal pathway in spinal ependymoma.It lays a theoretical foundation for the study of the molecular mechanism of ependymoma to be transformed into a new target for ependymoma treatment.Methods: In this study,patients with spinal ependyoma who underwent surgical treatment in the Neurosurgery Center of the First Affiliated Hospital of Xinjiang Medical University from February 2016 to July 2023 were included.The changes of modified McCormick scale(MMCS)before and after surgery were retrospectively analyzed.The correlation between clinical features,histopathology,preoperative imaging features and postoperative recurrence and recovery of spinal nerve function was investigated.Unsupervised clustering algorithm was used to distinguish different molecular subtypes of ependymal tumors.Gene set variation analysis(GSVA)was used to calculate TGF-β signal pathway-related scores in different ependymoma molecular subtypes.The survival rates of patients with different ependymoma subtypes were calculated by Kaplan-Meier analysis(KM).Linear models for microarray data were used to analyze the differential genes among different ependymoma subtypes.(Least absolute shrinkage and selection operator,least absolute shrinkage and selection operator,LASSO and COX proportional hazards model were used to construct a prognostic model of TGF-β signaling pathway related genes in ependymoma.Receiver operating characteristic curve(ROC)was used to evaluate the performance of prognostic prediction model.The correlation of key genes was investigated by Pearson correlation analysis.Tumor tissues of spinal ependyoma patients and normal tissues were collected as controls,which were divided into control group and case group.qRT-PCR and Western Blot were used to detect the expression levels of key genes in ependyoma tissues.Tumor cells were cultured from tumor tissues of spinal ependyoma patients,and FBN1 sh RNA was carried.The lentiviral gene of BMP4 sh RNA knocked out the expression of FBN1 or BMP4 protein,and the expression level of TGF-β gene was detected by qRT-PCR in tumor cells with low expression of BMP4 and FBN1.Transwell invasion assay and CCK-8 cell proliferation assay were used to lower the expression of FBN1 or BMP4 protein to detect the effect of tumor cell proliferation and invasion ability.The biological functions and regulatory mechanisms of related molecular biological signaling pathways in spinal ependyoma were verified by tissue sample verification and cell in vitro experiments.Results: There was no statistical significance in the recurrence of ependymoma patients with different postoperative pathological grades(P > 0.05).Compared with the immune function of patients with different postoperative pathological grades,there was a statistically significant difference in the positive ratio of CD57 among the three groups(P< 0.05).The positive ratio of GAFP,oligo-2,Neu-N,CD34,IDH-1,ki-67,EMA,PR,VIM,Syn,P53,D2-40,S-100,CD56,TIF-1,Cg A,AE1/AE3,CK,CEA,CD10 and nestin was not uniform among the three groups Statistical significance(P>0.05),Logistic regression results indicated that preoperative spinal edema(OR=13.914,B=2.633)was an independent risk factor for spinal cord dysfunction 3 months after surgery,while preoperative syringomyelia(OR=0.060,B=-2.813)was a protective factor.There was no statistical significance in age,sex,operation duration,tumor resection degree,pathological grade,or intraoperative spinal internal fixation(all P>0.05).Unsupervised clustering algorithm showed that there were two different molecular subtypes in ependymal tumors.GSVA and KM analysis showed that TGF-β signaling pathway scores and prognosis were significantly different in different subtypes of ependymomas.The results of gene differential expression analysis showed that TGF-β pathway related gene expression was significantly different in different subtypes of ependymomas.LASSO showed that BMP4,FBN1 and CDC20 were the key genes of prognosis prediction model.ROC analysis results showed that TGF-βsignaling pathway related gene model had high predictive performance.Pearson correlation analysis showed that FBN1,BMP4 and CDC20 were significantly correlated with their downstream genes in TGF-β signaling pathway.The results of q-PCR and Western Blot showed that compared with the control group,the expressions of FBN1 and BMP4 in spinal ependyma tissues were significantly up-regulated(P<0.01),while there was no significant difference in the expression of CDC20 in case group(P > 0.05).Knockdown of FBN1 or BMP4 not only down-regulated the expression of TGF-β gene in spinal ependymoma cells(P < 0.05),but also inhibited the proliferation and invasion ability of spinal ependymoma cells(P<0.01).Conclusion: The heterogeneous prognosis of ependymoma cannot be accurately predicted by current clinical and pathological molecular markers.The prognosis prediction model based on TGF-β pathway-related genes BMP4,FBN1 and CDC20 can effectively predict the survival of patients.Based on TGF-β pathway,this study investigated the influence of key molecules BMP4/FBN1 on the proliferation and invasiveness of spinal ependyoma,suggesting that FBN1,BMP4,CDC20 and TGFB1 in TGF-β signaling pathway play a role in regulating and activating signal channelation,and further promote the malignant progression of ependyoma.It is proposed that BMP4,FBN1,CDC20 and TGFB1 are the key biomarkers of ependymoma,which lays a theoretical foundation for the study of the molecular mechanism of spinal ependymoma to be transformed into a new target for ependymoma treatment.