Effect Of Phosphorylation Of Nrf2 Neh3 Domain On Chemoresistance Of GBC And Relative Mechanisms

Objectives1.Identification of new genes involved in oxidative stress associated with gallbladder cancers susceptibility and chemo-resistance.2.Investigation of how Nrf2,ROS and oxidative stress related genes interacted to influence chemo-resistance of gallbladder cancers.3.Development of new drugs which can enhancethe chemosensitivity of gallbladder cancerMethods1.Sorted SP cells with flow cytometry were detected ROS levels and apoptosis.Find genes with differential expression between SP cells and non-SP cells.2.RT-PCR detected expression of target genes in clinical samples.3.The expression of target genes were changed by usingplasmid overexpression or RNA interference.The expression level of m RNA and protein in cell lines were detected by RT-PCR or western blot.Effect of target genes expression on chemoresistance and its mechanism were explored.4.Explore upstream regulation mechanism of target genes with WB and RT-PCR and identify its stability regulation.5.Find special drug targeting on relative pathwayand explore its effect on chemoresistance of GBCResults1.Sorted SP cells showed stronger chemoresistance and lower ROS level.Nrf2 expression exhibited significant influence on chemoresistance of SP cells.Nrf2 and its downstream genes were selected as target genes in studying chemoresistance of GBC.2.RT-PCR test of clinical samples showed Nrf2 and its downstream target genes were upregulated in GBC.3.The expression levels of Nrf2 protein among GBC cells were reprogrammed.Nrf2 expression reduced ROS level and enhanced chemoresistance of GBC.4.Loss of Nrf2 Neh3 domain promoted nuclear location of Nrf2 and enhanced proliferation and chemoresistance of GBC cells5.Mutant sites were detected in Nrf2 Neh3 domain.GSK3β-Fyn pathway can regulate Nrf2 Neh3 domain.Conclusions1.Nrf2 and its downstream genes are associated with gallbladder cancer chemoresistnce.2.Loss of Nrf2 Neh3 domain promotedgallbladder cancer chemoresistance by upregulating Nrf2 and enhancing protein stability.3.Mutant sites in Nrf2 Neh3 domain promoted gallbladder cancer chemoresistance by regulating nuclear location of Nrf2 and enhancing protein stability.