| Background:Glioma is the most common malignant tumor in the central nervous system(CNS)of adults,and temozolomide(TMZ)chemoresistance is an important issue in the treatment of glioma.TMZ interferes with the DNA replication by promoting the formation of the O~6methylguanine adduct(O~6Me G)to play a cytotoxic role,while the DNA mismatch repair system can destroy the formation of the adducts,resulting in TMZ resistance.Therefore,it is of great interest to find a marker that can affect the DNA repair process.TSPAN7 was selected among the DEGs by bioinformatics analysis of RNA-sequencing data from gliomas that underwent TMZ treatment,and its prognostic value was validated in TCGA(The Cancer Genome Atlas,TCGA),CGGA(Chinese Glioma Genome Atlas,CGGA)and GEO(Gene Expression Omnibus,GEO)databases.TSPAN7,with low expression in high-grade gliomas and high expression in both IDH mutation and MGMT methylation groups,may serve as an important predictive factor for prognosis.In the field of the CNS,the research of TSPAN7 has focused on the neuronal synapses,as well as an involvement with actin in cytoskeletal regulation.However,the role of TSPAN7 in glioma has not been reported.Therefore,we focused on TSPAN7 to explore its biological function in glioma.ERS has been shown to be involved in chemoresistance in a variety of tumors including glioma,however,whether ERS is promoting or inhibiting for glioma remains controversial.Therefore,this study further explored the relationship and underlying mechanism with ERS through TSPAN7 downregulation mediated glioma malignant phenotype and TMZ chemoresistance.Objective:The aim of this study was to analyze the expression pattern of TSPAN7 in glioma in the TCGA,CGGA,and GEO databases.And the expression of TSPAN7 was detected in clinical samples of different grades of glioma and glioblastoma(GBM)cell lines U87 and LN229.Analyzing the effects of differential expression of TSPAN7 on the biological behavior of GBM cell lines U87 and LN229,including cell proliferation,cell cycle,invasion and migration,and EMT.Analyze the potential mechanism of TSPAN7 involvement in TMZ chemoresistance and validate in vivo.Methods:1.Analyzing the expression of TSPAN7 in glioma databases,clinical surgical samples and glioma cell linesRaw Affymetrix Hu Gene-1＿0-st-v1 Array profiles of TMZ resistant and sensitive glioblastoma clones were acquired from Array Express,the differential genes between TMZ resistance and sensitivity in gliomas were obtained,and TSPAN7 was selected as the research object of this study.Analyzing the expression and prognostic value of TSPAN7 in TCGA,CGGA,and GEO databases.Detect the expression level of TSPAN7 in different grades samples and glioma cell lines through q PCR and Western blot.2.Analyzing the effect of TSPAN7 on the proliferation,invasion,and migration in gliomaThe overexpression of TSPAN7 Lentivirus and si-TSPAN7 plasmids were constructed.Detect the effects of overexpression and knockdown TSPAN7 and explore proliferation of glioma cell lines by CCK-8 and Cloning formation assays.The wound-healing assay and Transwell assay were used to detect the impact of TSPAN7 on the invasion and migration for glioma cell lines.Flow cytometry was used to detect the impact of differential expression of TSPAN7 on cell cycle.The effect of TSPAN7 on the expression levels of epithelial mesenchymal transition(EMT)related biomarkers,including Vimentin,E-cadherin,N-Cadherin,ZO-1,was detected by Western blot in glioma cell lines,and the expression of E-cadherin and N-Cadherin was further verified by immunofluorescence.3.Exploring the role of TSPAN7 in TMZ chemoresistance during glioma treatmentAnalyzing the expression of TSPAN7 in the TMZ resistant and sensitive groups by processing the raw array profiles of GBM and RNA-seq data of U251 cell lines treated with TMZ.In TSPAN7 over expression and knockdown cell lines,the cell viability was detected using the CCK-8assay after TMZ treatment.To investigate the effect of TSPAN7 on cell apoptosis under TMZ treatment by detecting the immunofluorescence assay of cleaved caspase-3.γ-H2A immunofluorescence assay and Comet assay were performed to investigate the effect of TSPAN7 on DNA damage induced by TMZ.4.Exploring the intrinsic mechanism of TSPAN7 involved in TMZ chemoresistance during glioma treatment in vivo and in vitro.The DEGs related TSPAN7 were obtained by RNA sequencing(RNA-seq),and the upregulated and downregulated DEGs were subjected to gene ontology(GO)and Kyoto Encyclopedia of genes and genomes(KEGG)enrichment analysis.q PCR and Western blot were used to detect the m RNA expression levels of BIP,PERK,ATF6,IRE1,XBP1 and CHOP10and the protein expression levels of related pathways in the endoplasmic reticulum stress(ERS)pathway,and analyze the activation of the endoplasmic reticulum stress pathway.The binding of BIP with TSAPN7in the ERS pathway was analyzed by transfection of endoplasmic reticulum localization plasmid p Ds Red2-ER,immunofluorescence and co-immunoprecipitation experiments.Using q PCR and Western blot to detect the expression of DNA damage repair related enzymes MGMT,MPG,and Rad51.Results1.Bioinformatics analysis of the role of TSPAN7 in glioma.(1)As the increasing of WHO grade,the expression level of TSPAN7 gradually decreases.(2)The expression level of TSPAN7 is significantly correlated with IDH mutation,1p19q deletion,MGMT promoter methylation,and GBM molecular subtypes.(3)The expression level of TSPAN7 is associated with the prognosis of glioma patients,and the higher the expression of TSPAN7,the better the prognosis of patients.2.Study on the role of TSPAN7 in the proliferation,invasion and migration in glioma(1)Overexpression of TSPAN7 significantly inhibits the proliferation,invasion,and migration of glioma cells,knockdown of TSPAN7 significantly promotes cell proliferation,invasion,and migration,and affects the expression of EMT related proteins.(2)Overexpression of TSPAN7 promotes cell cycle arrest in the S phase,while knocking down TSPAN7 can reduce the proportion of S phase cells.3.Study on the TMZ chemoresistance of TSPAN7 in glioma.(1)Overexpression of TSPAN7 reduce the tolerance and promote cell apoptosis and DNA damage induced by TMZ in glioma,while knocking down TSPAN7 increase the chemoresistance and inhibit cell apoptosis and DNA damage.4.Study on the mechanism of ERS involved in the chemoresistance of glioblastoma(1)Overexpression of TSPAN7 activates endoplasmic reticulum stress related pathways.(2)Overexpression of TSPAN7,the nuclear localization of ATF6significantly increased.(3)After overexpression of TSPAN7,the binding of BIP to TSPAN7increased,while the binding of ATF6 to BIP decreased significantly.(4)After overexpression of TSPAN7,the expression level of DNA damage repair related enzyme MGMT decreased.(5)In situ tumor formation experiments in nude mice have confirmed that overexpression of TSPAN7 can significantly inhibit tumor growth,increase tumor sensitivity to TMZ,and improve the survival.Conclusion(1)The expression of TSPAN7 gradually decreases as the increasing of WHO grade.The expression level of TSPAN7 is significantly correlated with IDH,1p19q,and MGMT promoter methylation status.The higher the expression level of TSPAN7,the better the prognosis of patients.(2)TSPAN7 can significantly affect the proliferation,invasion,migration,EMT,and cell cycle of glioma cells.(3)Overexpression of TSPAN7 significantly reduce the chemoresistance of glioma cells to TMZ.In this process,competitive binding of TSPAN7 and BIP to activate the ERS pathway,thereby down-regulating the expression of DNA damage repair related enzyme MGMT.Figures 26,Tables 19,Reference 161... |
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