The Study Of Effects Of TCIRG1 Expression On The Malignant Phenotypes And Prognoses Of Glioma As Well As Its Mechanism In Regulating Malignant Biological Behaviors

Objective: High-grade gliomas represented by glioblastoma(GBM)have caused a great danger to the human body,and standardized surgical treatment combined with post-operative radio-and chemotherapy still leads to a less than 10% of 5-year survival for GBM,which is a huge threat to human health.With the advancement of molecular biology technology,the traditional histopathology-based diagnostic criteria are undergoing a great challenge.The change of using molecular pathology as the basis for diagnosis is happening.With the publication of the 2016 classification of central nervous system(CNS)tumors,molecular pathology diagnosis of glioma has played an important role in guiding clinical neurosurgical practice,despite which progress in the treatment of glioma remains very limited,and the exploration of molecular mechanisms and therapeutic targets for glioma pathogenesis have never ceased.Vacuolar ATPase(V-ATPase)is a class of macromolecular complex whose role in tumors has been widely explored,but studies on the roles and mechanisms of its specific subunits in glioma are still limited.TCIRG1 is the V0a3 subunit of V-ATPase,which is involved in mediating the localized distribution of V-ATPase on tumor cell membranes and the regulation of a series of malignant biological behaviors,but its role in glioma has not been reported.In this paper,we initially elucidated the relationship between this molecule and the malignant phenotypes of glioma,and further explored the regulation of malignant biological behaviors of glioma and the related mechanisms of this molecule.Methods: 1.Bioinformatics analysis was performed to obtain transcriptional data from the TCGA,CGGA and GEO databases of glioma patients,to initially clarify the expression of TCIRG1 gene in different glioma pathological grades,tissue types and clinical and molecular phenotypes,and to elucidate the impact of its expression on the overall survival(OS)of glioma patients,and to determine the independent role of TCIRG1 in glioma prognosis by multivariate Cox regression analysis.2.The expression of TCIRG1 was further verified using clinical samples and glioma cell lines.3.Stable glioma cell lines with knockdown and overexpression of TCIRG1 were constructed.4.CCK-8,EdU,clone formation assay,cell scratch assay,and Transwell stromal gum invasion assays were used to determine the proliferation,migration,and invasive ability of glioma cells after knockdown and overexpression of TCIRG1.5.The effect of silencing and overexpression of TCIRG1 on apoptosis of glioma cells was detected by flow cytometry and Tunel assay,and the effects of interference with TCIRG1 expression on apoptosis-related protein expression was detected by Western Blot(WB).6.The single sample gene set enrichment analysis(ssGSEA)was used to calculate the enrichment scores of hypoxia,angiogenesis and epithelial-mesenchymal transition(EMT)in glioma,and the close correlation between TCIRG1 expression and the above scores was initially clarified by correlation analysis.7.The effects of TCIRG1 on glioma-related biological processes were investigated by constructing hypoxic cell culture environment.To detect the effect of hypoxia on the expression of TCIRG1 with EGFR and HIF1 A,the co-expression of TCIRG1 with EGFR and HIF1 A was detected by co-immunoprecipitation(CO-IP)assay.WB and immunofluorescence experiments were used to verify the changes of EMT markers in glioma cell lines after interfering with TCIRG1 expression,and the effects of TCIRG1 expression on glioma EMT process through EGFR/HIF1 A axis were investigated by adding EGFR inhibitor and HIF1 A overexpression.8.WB experiments were performed to verify the effects of interfering TCIRG1 expression under hypoxic environment on PI3K/Akt/mTOR pathway.After using EGFR inhibitor,we verified the effect of TCIRG1 on the malignant biological behaviors of glioma through regulating the expression of EGFR and then affecting PI3K/Akt/mTOR pathway.9.Exploring the effects of TCIRG1 expression on the malignant biological behaviors of glioma through the PI3K/Akt/mTOR pathway by adding pathway inhibitor.10.Subcutaneous tumorigenesis assay in nude mice was performed to detect changes in tumorigenic ability of tumor cells in vivo after interference with TCIRG1 expression.And tumor specimens were taken to explore the changes of EMT-related indexes,Ki-67 and VE-cadherin in tumor tissues by immunohistochemistry and WB assays.11.Graphpad Prism 7.0 and R software(version3.6.3)were used for graphing and statistics,and t-test or ANOVA analysis were used for comparison between groups,Log-rank test was used for survival analysis,and correlation studies using Pearson or Spearman correlation coefficient analysis,with P < 0.05 in the statistical results as a statistically significant difference.Results: 1.The sequencing data from the TCGA,CGGA and GEO databases based on bioinformatics analysis showed that TCIRG1 expression was higher in gliomas than in normal brain tissues and the highest was found in GBM(WHO grade IV).High TCIRG1 expression could reflect different malignant phenotypes of gliomas,with IDH wild type,1p19 q non co-deletion type,older age(>60 years)and MGMT non-methylation group had significantly higher TCIRG1 expression in gliomas.In addition,TCIRG1 was highly expressed in mesenchymal GBM and may be a valid indicator for the diagnosis of mesenchymal GBM.2.Higher expression of TCIRG1 was verified in human WHO IV grade glioma tissues by immunohistochemical experiments.Meanwhile,we verified that TCIRG1 expression was higher in glioma cell lines than in normal astrocyte line using RT-PCR and WB assays.3.TCIRG1 high expression can effectively reflect the prognosis of glioma patients,and its high expression suggests poor prognosis of different clinical and molecular phenotypes of glioma patients,as well as being an important indicator of poor prognosis after glioma chemotherapy and radiotherapy.4.Consensus clustering analysis showed that the transcriptional expression of TCIRG1 combined with other four V-ATPase coding genes(ATP6AP1,ATP6AP2,ATP6V1C2 and ATP6V1G2)could classify gliomas into 2 subtypes with different prognosis and clinical phenotypes,among which the patients in C2 subtype had a worse prognosis with activation of tumorand immune-associated pathways.Gene ontology(GO)and KEGG pathway analysis suggest that TCIRG1 may be involved in the regulation of multiple biological pathways in gliomas.5.The interference of TCIRG1 expression significantly affected the proliferation,migration,and invasion of glioma cell lines,as confirmed by CCK-8,clone formation,EdU,wound healing,and Transwell assays.In vivo tumorigenesis assays in nude mices also confirmed that high expression of TCIRG1 promoted the proliferation ability of gliomas.6.Tunel and flow cytometry assays showed that interference with TCIRG1 expression affected the apoptotic processes of glioma,and silencing TCIRG1 expression increased the expression of apoptosis-promoting proteins Caspase-3,PARP1,and Bax,while the expression of apoptosis-inhibiting protein Bcl-2 decreased.Overexpression of TCIRG1 down-regulated the expression of Caspase-3,PARP1,and Bax,while Bcl-2 expression increased.7.Ivy GAP analysis showed that the transcript levels of TCIRG1 varied in different anatomic regions of the GBM,with significantly higher expression in the area of hyperplastic blood vessels in tumor cellular and microvascular proliferative regions.The GSEA showed that glioma patients with high TCIRG1 expression were associated with activation of angiogenesis,epithelial mesenchymal transition(EMT),and hypoxia pathways.The enrichment scores of angiogenesis,epithelial mesenchymal transition,and hypoxia processes in glioma patients were constructed by ssGSEA,and TCIRG1 expression showed a significant positive correlation with the above scores.8.Interference with TCIRG1 expression in hypoxic environment significantly affected the EMT process in glioma.By WB and immunofluorescence experiments,knockdown of TCIRG1 resulted in upregulation of EMT-related marker protein E-cadherin expression,while N-cadherin,Vimentin,and ZEB1 expression were downregulated;conversely,E-cadherin expression was downregulated,while N-cadherin,Vimentin,and ZEB1 expression were upregulated.In vivo experiments,the results were also consistent with the above results.9.Under hypoxia environment,overexpression of TCIRG1 upregulated the expression of EGFR and HIF1 A,and conversely,knockdown of TCIRG1 downregulated the expression of both;the CO-IP experiment confirmed that TCIRG1 and EGFR had a protein interaction relationship,and EGFR inhibitor could block the upregulation of HIF1 A after TCIRG1 overexpression.10.Under hypoxic environment,TCIRG1 regulates the EMT process in glioma cell lines through the EGFR/HIF1 A axis;TCIRG1 regulates the expression of key proteins of the PI3K/Akt/mTOR pathway through EGFR.11.TCIRG1 regulates the proliferation,migration,and apoptotic processes of glioma cell line through the PI3K/Akt/mTOR signaling pathway under hypoxic environment.12.Down-regulation of TCIRG1 affects the expression of CD31,a marker molecule of glioma angiogenesis,and the expression of TCIRG1 is significantly positively correlated with vascular mimetic markers.TCIRG1 regulates the expression of VE-cadherin,a microvascular mimetic marker of glioma,through HIF1 A.Conclusions: 1.TCIRG1 is highly expressed in human brain glioma tissue and predicts tumor malignancy,and may be a specific marker for the mesenchymal subtype of glioblastoma(GBM),and its high expression suggests poor prognosis for glioma patients.2.TCIRG1 high expression promotes the ability of human brain glioma cells to proliferate,migrate,and invade,and affects the occurrence of glioma apoptosis.3.Under hypoxic environment,TCIRG1 can regulate the EMT process and vascular mimicry formation in glioma through EGFR/HIF1 A axis.4.Under hypoxic conditions,TCIRG1 regulates the expression of key proteins in the PI3K/Akt/mTOR signaling pathway through EGFR,and affects the malignant biological behaviors of human glioma through PI3K/Akt/mTOR pathway.