Study On The Correlation Between Radiation Resistance Molecular Type Of Glioblastoma Cell Line And Activation Of NF-κB And EGFR

Background and Objective:Glioblastoma multiforme(GBM)is a malignant tumor of the central nervous system.Radiotherapy is its conventional treatment.However,GBM is prone to radiotherapy resistance,which greatly restricts the efficacy of radiotherapy.After radiation,the EGFR pathway and the NF-κB pathway are activated together to promote the repair of DNA damage in GBM and cause radiotherapy resistance.To investigate the radiotherapy prognosis of clinical samples with different molecular types of GBM and the radiation resistance of the corresponding cell lines.At the same time,the time-dependent changes of DNA breakage,NF-κ B effector molecule(RELA/p65)and EGFR activation after radiation were studied,so as to provide a preliminary basis for the best clinical use of radiosensitizers(NF-κ B inhibitor,EGFR inhibitor).Methods:1.The expression of P53,IDH1,PTEN and MGMT in GBM clinical specimens and the prognosis of radiotherapy were analyzed by using Chinese glioma atlas database(Chinese Glioma Genome Atlas,CGGA).2.GBM cell lines(U87MG,U251 MG,U118MG)were selected as the research objects.The mutations of P53,IDH1 and PTEN genes were detected by exon sequencing,and the expression of MGMT was detected by Western blotting to determine their molecular typing.3.After exposing to different doses of radiation(2Gy,5Gy,10Gy),the morphological changes of three cell lines were observed,the level of apoptosis and the ability of cell proliferation were detected,and the radiation resistance of GBM cell lines with different molecular types was analyzed.4.Western blotting was used to detect the activation of DNA damage markers(γH2A.X),RELA/p65 and EGFR after different doses of radiation(2Gy,5Gy),and to analyze the time dependence and correlation of DNA damage,NF-κ B and EGFR signal pathway activation..Results:1.CGGA database analysis shows: the mutation rates of P53,IDH1,and PTEN in primary GBM are 48%,19%,and 17%,respectively,and the mutation rates in recurrent GBM are 52%,25%,and 12%,respectively.P53 wild-type(P53wt),IDH1 mutant(IDH1mt),PTEN wild-type(PTENwt),and glioma patients with low expression of MGMT who received radiotherapy have a longer overall survival.2.U87 MG has P53-exon-2,P53-exon-3,P53-exon-4,IDH1-exon-189,IDH1-exon-201 and PTEN-exon-9 mutations,and low expression of MGMT.U251 MG has P53-exon-2,P53-exon-3,P53-exon-4,P53-exon-7,PTEN-exon-7 homozygous insertion TT and PTEN-exon-9 mutations,no IDH1 mutations,and low Express MGMT.U118 MG has mutations of P53-exon-2,P53-exon-3,P53-exon-4,P53-exon-5,IDH1-exon-201 and PTEN-exon-9,and highly expresses MGMT.3.After 2Gy,5Gy,and 10 Gy radiation,the cytoplasm of the non-irradiated group is transparent and the cell membrane is intact.The number of cells in the radiation group decreased,and the cytoplasm was vacuolated.Apoptosis rate change value: U87MG>U118MG>U251MG(p<0.05).The survival score at each dose: U251MG>U118MG>U87MG(p<0.05).Prompt radiation resistance:U251MG(P53mt/IDH1wt/PTENmt/MGMTpm)>U118MG(P53mt/IDH1mt/PTENmt/MGMTpu)>U87MG(P53mt/IDH1mt/PTENmt/MGMTpm)4.Changes in γH2A.X: At 2Gy,U87MG(started at 1h and reached the peak,recovered at 48h)>U118MG(started at 1h and reached the peak,recovered at 48h)>U251MG(started at 1h and reached the peak,recovered at 48h).At 5Gy,U87MG(started at 1h and reached the peak,48 h did not recover)> U118MG)(started at 1h and reached the peak,48 h recovered)>U251MG(started at 1h and reached the peak,48 h recovered).The severity of DNA fragmentation after radiation: U87MG(P53mt/IDH1mt/PTENmt/MGMTpm)>U118MG(P53mt/IDH1mt/PTENmt/MGMTpu)> U251MG(P53mt/IDH1wt/PTENmt/MGMTpm).5.NF-κB activation: At 2Gy,U87MG(started at 3h and reached the peak,48 h did not recover)> U118MG(started and reached the peak at 3h,recovered at 48h)> U251MG(started and reached the peak at 3h,recovered at 48h).At 5Gy,U87MG(started at 1h,reached the peak at 3h,did not recover at 48h)> U118MG(started at 1h,reached the peak at 6h,recovered at 48h)> U251MG(started at 1h and reached the peak,recovered at 24h).The duration of NF-κB activation is: U87MG(P53mt/IDH1mt/PTENmt/MGMTpm)>U118MG(P53mt/IDH1mt/PTENmt/MGMTpu)>U251MG(P53mt/IDH1wt/PTENmt/MGMTpm).6.EGFR activation: at 2Gy,U87MG(starts at 3h,reaches the peak at 6h,does not recover at 48h)> U118MG(starts at 3h,reaches the peak at 6h,and has recovered at 48h)>U251MG(starts at 3h,reaches the peak at 6h,and has recovered at 48h).At 5Gy,U87MG(started at 1h,peaked at 12 h,and did not recover at 48h)>U118MG(started at 1h,peaked at 6h,and did not recover at 48h)>U251MG(started at 1h,peaked at 3h,and did not recover at 48h).The duration of EGFR activation is: U87MG(P53mt/IDH1mt/PTENmt/MGMTpm)>U118MG(P53mt/IDH1mt/PTENmt/MGMTpu)> U251MG(P53mt/IDH1wt/PTENmt/MGMTpm)Conclusions:1.The molecular classification of GBM is related to the prognosis of radiotherapy and resistance to radiotherapy.2.GBM with high expression of P53 mt,IDH1wt,PTENmt,and MGMT is more resistant to radiotherapy,and the overall survival of patients is shorter.3.DNA fragmentation and activation of NF-κB pathway and EGFR pathway are time-dependent after GBM radiation in different molecular types.