Molecular Mechanism Of CREB In Glioma Multi-drugresistant

Objective:To analyze whether the CREB is involved in glioma cell line U251 multi-resistant formation and possible mechanism.To find out a new molecular target which can reversing MDR of glioblastoma.Methods:Immunohistochemical method to detect and compare the different level of glioma cells CREB expression.Cultivate stable glioma U251 TMZ-resistant cell lines:U251/TR,CCK 8 to detect cell resistance index,specific knock CREB gene out of U251/TR,Flow Cytometric Method(FCM)to detect the cell apoptosis between U251/TR and U251 under the action of temozolomide(TMZ)?Use Crispr/cas9 to knockout CREB gene of U251 / TR to receive U251 / RC.RT-pcr and Western to detect ABCG2 gene,MGMT,MRP and P-gp gene expression levels of U251/TR and U251/RC.Results:This study successfully cultivate a stable resistance from cell line U251 cell line U251 / TR.On TMZ induced initial dose 5 mu mol/L,final dose 400 mu mol/L DMEM medium,U251 IC50 for mu(41.36 + 1.61)mol/L,U251 / TR IC50 for mu(293.09 + 2.46)mol/L,U251 / TR is U251 seven times.The Crispr/cas9 specificity knockout U251 / TR CREB gene cell line U251 / RC,TMZ intervention dose 100 mu mol/L medium,apoptosis of U251 / TRC level is much higher than U251 / TR.And rt-pcr and Western,according to the results of U251 / RC ABCG2,MGMT,MRP and P-gp expression levels were significantly reduced.Conclusions:The CREB gene regulates downstream ABCG2,MGMT,the expression of MRP and P-gp transporter?s levels involved in drugs,drug targets,cell apoptosis,cell repair,such as process,affecting cell multi-resistant to reverse.This provides a new direction to fundamentally solve the current clinical glioma cure and postoperative recurrence of dual problem.