| Part 1 Exploring potential therapeutic targets for high-grade meningiomas based on bioinformatics technologyObjective Bioinformatics was used to explore the differentially expressed genes between WHO grade I meningioma and high-grade meningioma(HGM).Survival analysis revealed the biomarkers affecting the poor prognosis of HGM and explored its potential therapeutic targets.Methods According to the GEO2 R function of GEO website,WHO grade I meningiomas and high-grade meningiomas(HGMs)were set as control group and experimental group respectively.The DEGs in the databases of two independent research centers were analyzed.FC > 1.5 and adj.P.value < 0.05 were taken as the criteria to further screen out the differentially expressed genes(DEGs).KEGG pathway annotation and GO enrichment analysis were performed on DEGs.The protein-protein interaction(PPI)between DEGs encoded proteins was analyzed by string online software.The PPI network was constructed with combined score> 0.4 as the screening condition.The results of K-means clustering were exported to Cytoscape software.The top 10 gene in PPI network is selected as hub gene based on degree algorithm by using Cyto Hubba plug-in.According to the median value of hub gene expression,the patients were divided into high and low expression groups.The Kaplan-Meier curve was drawn using the follow-up information of the database.The tumor grade,gender,age and hub gene expression were included in the multivariate Cox regression analysis,and the key genes that can independently predict the prognosis of HGMs were obtained.Result There were significant differences in the expression of DEGs between grade I and HGMs,of which 51 were up-regulated and 73 were down-regulated.510 pathways with statistical differences were obtained through GO enrichment.After inputting DEGs into string database,K-means clustering analysis showed that CKS2,GINS2,UBE2 T,MCM2,TPX2,PTTG1,NCAPG,CCNB1,BUB1,CKD1,AURKA,CEP55 and other genes clustered in the same region,which may have interaction relationship.Further quantitative analysis using the degree algorithm,the top 10 gene was obtained.The K-M curve of TOP10 gene was drawn one by one and log rank test was performed.The expression of ASPM,MELK,AURKA,KIAA0101,NCAPG,CCNB1 and BUB1 was closely related to the prognosis and recurrence of high-grade meningioma.Cox regression analysis showed that the expression of ASPM,CCNB1 and BUB1 was negatively correlated with PFS,which was an independent risk factor for recurrence of high-grade meningioma.Conclusion ASPM,CCNB1 and BUB1 are potential key genes that can independently predict the recurrence risk of HGMs,and can be used as therapeutic targets for HGMs in the future.Part 2 The mutual relationship between the expression of key genes and the prognosis of high-grade meningioma verified by tissue microarrayObjective To detect the expression and clinical significance of ASPM,CCNB1 and BUB1 in HGMs by tissue microarray and immunohistochemistry,and to provide new ideas for the diagnosis and treatment of HGMs.Methods The information of patients with meningioma treated and operated on in Shanghai Changzheng Hospital between 2000 to 2020 was retrospectively collected.The tissue samples confirmed by pathology as WHO grade II and III meningiomas were fixed and embedded into TMA together with the selected dura and WHO grade I meningiomas,and the tissue chip was well prepared.The expression levels of BUB1、CCNB1、ASPM in tissue microarray were detected by immunohistochemical technique,and the results were interpreted by pathological experts.The clinical data and follow-up data of patients corresponding to the chip was collected and sort out.The relationship between gene expression level and tumor recurrence was analyzed,and its effect on the development of HGMs was obtained.Result From 2000 to 2020,271 patients with WHO grade II and III meningiomas accepted surgical treatment in the neurosurgery of Changzheng Hospital,accounting for 10.1%(271/2688)of all the meningiomas treated in the same period.103 males and 168 females were included,with a male to female ratio of 1:1.63.There were 174 cases of primary cases and 97 of recurrence,with ratio of 1:0.56.The postoperative histopathological results showed that there were 216 cases of WHO grade II,accounting for 89.8%,and 52 cases of WHO grade III,accounting for 19.2%.A total of 96 specimens were included in the tissue microarray,including 9 cases of normal dura in the blank control group,and 15 of grade I,63 of grade II and 9 of grade III in the experimental group.The positive rate and total integral value of ASPM,CCNB1 and BUB1 genes in HGMs in the experimental group were higher than those in WHO grade I meningiomas in the control group significantly.There were 49 HGMs got followed up in all cases of tissue microarray,and 23 cases recurred after operation,while 14 cases accepted adjuvant radiotherapy.The follow-up time was 46.8±25.9(15-91)months.The expression levels of BUB1 and CCNB1 had a significant effect on PFS in HGMs,and the PFS in patients with high expression group was lower than that in patients with low expression group significantly.Age,sex,tumor size,tumor nature,tumor location,resection,postoperative adjuvant radiotherapy and other factors that may affect the prognosis were included in the Cox risk regression model for further analysis.It can be found that the high expression of Bub1 and ccnb1 is an independent risk factor for the prognosis of HGMs.Conclusion The expression levels of Bub1 and ccnb1 proteins were significantly higher in HGMs.Their expression levels were closely related to the poor prognosis of patients with HGMs,and patients with high expression were more likely to recurrence.Part 3Objective To clarify the function of BUB1 in the occurrence and development of malignant meningioma.Methods A specific lentivirus vector targeting Bub1 overexpression was designed and synthesized,and the Bub1 overexpressed malignant meningioma cell line was constructed by plasmid transfection.CCK-8,scratch and Transwell experiment was used to detect the changes of cell proliferation,migration and invasion.And the changes of cell cycle was detected by flow cytometry.Result After overexpressing of BUB1,the proliferation ability of IOMM-Lee cell line was significantly higher than that of NC group,and the distance came to largest on the fourth day.The migration ability was significantly higher than that of the NC group as well,and the degree became largest on the second day.The migration ability of the OE group increased by 32.3%.The results of cell cycle detection by flow cytometry showed that the cells in OE group had a certain degree of G1 checkpoint block.Compared with NC group,the fraction of G1 phase cells in OE group increased by 17.3%,and the proportion distribution of S phase and G2/M phase cells decreased by 30.2% and 24.1%.After the inhibition of BAY 181603,the proliferation ability of iomm Lee cell line was significantly lower than that of NC group,the difference was significant after 72 hours,and the distance was the largest after 96 hours;The migration ability decreased significantly compared with NC group,and the difference reached the maximum on the second day.The migration ability of BAY group decreased by 21.4%.Conclusion Overexpression of BUB1 can promote the proliferation,invasion and migration of malignant meningioma cells.The inhibitor BAY 181603 inhibits the malignant proliferation of malignant meningioma cells by targeting Bub1. |
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