Discovery And Study Of Key Proteins Of Temozolomide Resistance Mechanism In Cerebral Glioma

Glioblastoma(GBM)is an aggressive malignant brain tumor with a median survival of 12-15 months.The conventional treatment is surgical resection combined with radiotherapy and Temozolomide(TMZ)adjuvant chemotherapy.The intrinsic and acquired drug resistance of GBM to TMZ are the main obstacles in the treatment of GBM.The established mechanisms include the blood-brain barrier against the action of tumor targeting drugs,but the mechanism of drug resistance is still not very clear.Several studies have shown that O6-methylguanine methyltransferase(MGMT)can directly repair tumor gene damage induced by TMZ,which is an important cause of TMZ drug resistance.However,it was recently found that some GBM patients with TMZ resistance showed loss of MGMT activity.Therefore,there is an urgent need to identify TMZ resistance mechanisms independent of MGMT and develop alternative chemotherapy strategies for GBM patients.Based on CPTAC database,CtIP and PAXX genes with high change frequency were screened out in glioma in this study.CGGA database was used to analyze the correlation between its expression level and survival and prognosis of patients.The results showed that low m RNA level and high methylation level were beneficial to survival and prognosis of patients,that is,inhibition of RBBP8 and PAXX genes was beneficial to survival of patients.Spearman coefficient was used to calculate the correlation,and co-expressed genes were screened.KEGG was used to analyze the enrichment signal pathways,and it was found that they were mainly enriched in the pathways of metabolism,apoptosis and DNA damage repair,which laid a foundation for subsequent experiments to clarify the potential value of CtIP and PAXX genes in the treatment of glioma.At the same time,we perform quantum chemical calculation for TMZ and its active drug MTIC in human body,describe its chemical properties from HOMO-LUMO orbitals(Highest Occupied Molecular Orbital-Lowest Unoccupied Molecular Orbital),ELF(electron localization function),ALIE(mean local ionization energy),and explore its potential drug function based on target prediction and molecular docking.The discovery of its potential to inhibit HR repair,JNK pathway and NFκB pathway contributes to a deeper understanding of this drug and provides clues for the subsequent development of novel anti-glioma drugs.Previous published studies have shown that DNA damage repair is an important mechanism contributing to TMZ drug resistance.Combined with biological interestology analysis,we believe that CtIP and PAXX,the key factors of DNA damage repair,may be potential targets for improving TMZ-resistant GBM chemotherapy.Clinical use of TMZ can affect the occurrence of autophagy,so as to escape death.The successful escape of cell death is another influencing factor of TMZ drug resistance.Therefore,influencing the way of death of GBM is also an important way to lead to its resistance to TMZ.Further understanding how TMZ affects the death of GBM and the changes of the way of death after TMZ resistance is of great significance for a profound understanding of the process and mechanism of TMZ resistance of GBM.In this paper,TMZ-resistant U87 cell line(U87-TR)was constructed by increasing drug concentration method.Subsequently,based on the important role of CtIP and PAXX in DNA repair,CRISPR/Cas9 technology was used to knock out CtIP and PAXX respectively.The role of CtIP and PAXX in reversing TMZ resistance was investigated by analyzing protein expression,cell growth,clonal formation and tumor formation in nude mice.The molecular mechanisms of CtIP and PAXX in TMZ resistance were further investigated,including protein interaction,homologous recombination(HR),non-homologous terminal junction(NHEJ)and base resected repair(BER).The results showed that the expressions of CtIP and PAXX in U87-TR cells were significantly higher than those in wild-type U87(U87-WT)cells,and TMZ could induce the up-regulation of CtIP and PAXX in U87-WT cells.In CtIP knockout(CtIP-/-)U87 cells,the efficiency of NHEJ decreased significantly.In PAXX knockout(PAXX-/-)U87 cells,BER efficiency decreased significantly and iron death increased.Iron death plays a key role in the occurrence and treatment of tumors,especially its role in tumor drug resistance mechanism has attracted increasing attention in recent years.These experimental results suggest that CtIP and PAXX play an important role in drug resistance mechanism.Further studies revealed the interaction between CtIP and DNA Ligase IV and PAXX and DNA polβ.Cell growth experiments showed that inhibition of CtIP and PAXX expression could reverse TMZ resistance in glioma cells.The purpose of this study is to explore the molecular mechanism of action and potential targets of TMZ resistance in GBM.The key regulatory proteins CtIP and PAXX of TMZ resistance in GBM have been discovered,and the molecular mechanism of TMZ resistance in GBM has been clarified,providing a new potential target for the treatment of GBM,which is expected to enhance the efficacy of TMZ and reverse the drug resistance.To provide new methods and options for improving the prognosis of GBM patients.Future studies will further explore the potential diagnostic and therapeutic value of abnormal expression of CtIP,PAXX and SLC7A11 in GBM,as well as the specific role and regulatory mechanisms of iron death in GBM resistance.