Screening And Identification Of Temozolomide Resistance Gene In Glioblastoma Based On CRISPR/Cas9 Library

Background:Glioblastoma multiforme（GBM）is a highly aggressive and fatal malignancy of the central nervous system.Temozolomide（TMZ）is the first-line chemotherapeutic drug to treat GBM,although drug resistance remains challenging.To date,MGMT promoter hypomethylation explains part of TMZ resistance cases.However,this is not the only factor,and exploring more molecular mechanisms of TMZ resistance would help to optimize GBM treatment.Objective:The purpose of this study was to utilize the whole human Genome-scale CRISPR-Cas9 knockout（GeCKO）library to screen for novel genes involved in TMZ resistance and explore their functions in GBM cell lines.Methods:The GeCKO library was transferred into U251 and U87cells using lentivirus.After 72 h treatment with TMZ,DNA was extracted from the surviving cells for high-throughput sequencing,and the candidate genes were obtained by intersecting the sequenced gene sets.After modifying the expression of candidate genes using the constructed siRNA or plasmid,then candidate genes were filtrated by CCK-8 analysis and validated by flow cytometry apoptosis.Then,tumor samples from stageⅣGBM patients were used to assess the correlation between the validated genes and progression-free survival（PFS）of the patients.On the other hand,the role of TMZ resistance-related genes in GBM tumorigenesis was explored in U251 and U87 cell lines.The EdU assay,cell proliferation curve assay and plate cloning experiment were carried out to explore the influence of genes on proliferation ability.Finally,the scratch experiment and transwell assay were conducted to explore the influence of genes on migration and invasion ability.Results:Seven candidate genes including USP17L20,CD99L2,IL2RG,STEAP4,ALKBH3,EDARADD,and PPAPDC3 were identified.Among them,the CD99L2 gene had been confirmed to contribute to TMZ resistance.In U251 and U87 cells treated with TMZ,the knockdown of CD99L2 improved IC₅₀ by 41.96%and 54.97%compared with the NC groups,respectively.The overexpression of CD99L2reduced IC₅₀ by 48.42%and 41.84%compared with the vector groups.Moreover,the patients with high-expressed CD99L2 were associated with longer PFS（median PFS:7.87 months vs.2.7 months,P=0.0003）.In addition,CD99L2 was lower expressed in GBM than in normal brain tissue or normal glial cells.The overexpression of CD99L2 also inhibited GBM cell proliferation,aggression,vice versa.Conclusion:This study found that knocking down CD99L2 could contribute to TMZ-resistance and poor prognosis in GBM.CD99L2 could inhibit cell proliferation,migration and invasion.