To Investigate And Analyze The Mechanism Of EZH2 Regulating Hedgehog Signaling Pathway In Non-small Cell Lung Cancer

Background: Non-small cell lung cancer(NSCLC)is a malignant tumor disease with the highest incidence and mortality rate in the world.Although the current methods of diagnosis and treatment continue to develop,the 5-year survival rate is only 19.8%.The pathogenetic process of NSCLC involves a variety of molecular mechanisms and multiple environmental risk factors.By studying related mechanisms of relevant molecular pathways,it is helpful to define the pathogenesis and its process,and provide a theoretical basis for seeking new diagnostic and therapeutic targets and treatment pathways.Studies suggested that EZH2 mediates the regulation of multiple signaling pathways by silencing the methylation of target genes through catalytic action.In addition,it is involved in the growth,metastasis,drug resistance and other processes of NSCLC.And the high expression of EZH2 in NSCLC patients is closely related to poor prognosis.Prior to this,targeting different types of NSCLC cell strains,chromatin immunoprecipitation-promoter chip(CHIP-on-chip)technique was adopted,by which it was found that EZH2 was bound to Cul4 A gene promoter region,suggesting that Cul4 A genes may be subject to targeted regulation of EZH2.Besides,studies have shown that Cul4 A,a core member of E3 ubiquitin ligase,may regulate the m TOR pathway through ubiquitination.Other reports indicated that the activation of m TOR may cause high expression of Gli.Therefore,it is speculated that EZH2 may regulate Hedgehog-Gli pathway through the m TOR pathway,i.e.whether the EZH2-m TOR-Gli pathway exists.Objective:1.To confirm that EZH2 gene is involved in the activation of Hedgelog-Gli pathway in non-small cell lung cancer.2.To explore the mechanism of EZH2 in non-small cell lung cancer(NSCLC)and provide evidence for exploring potential molecular targets for diagnosis and treatment of NSCLC.Method:1.Exogenous EZH2 was subject to plasmid transfection in H226 and H441 with low expression of EZH2,and the changes of m RNA expression in EZH2,P21,N-Myc,BCL1 and GLi1 m RNA was detected by Real-time PCR after EZH2 up-regulation.2.Exogenous EZH2 was subject to plasmid transfection in H226 and H441 with low expression of EZH2,and m TOR inhibitor rapamycin was used on cells.Then the changes of m RNA expression in P21,N-Myc,BCL1 and Gli1 were detected by Real-time PCR.3.Exogenous EZH2 was subject to plasmid transfection in H226 and H441 with low expression of EZH2,and protein expression changes in P21,N-Myc,BCL1 and GLi1 was detected by Western blot after up-regulation of EZH2.4.Exogenous EZH2 was subject to plasmid transfection in H226 and H441 with low expression of EZH2,and m TOR inhibitor rapamycin was used on cells.Then the changes of protein expression in P21,N-Myc,BCL1 and Gli1 were detected by Western blot.Result:1.After 48 hours of transfection of exogenous EZH2 gene in H226 and H441 cells with low expression of EZH2,Real-time PCR and Weztern blot results showed that the expression of P21 in H226 and H441 cells with high expression of EZH2 decreased,the expression of N-Myc did not change significantly,while the expression of BCL1 and Gli1 increased.2.After 48 hours of transfection of exogenous EZH2 gene in H226 and H441 cells with low expression of EZH2,rapamycin treatment could re-up-regulate the expression of P21,down-regulate the expression of N-Myc,down-regulate the expression of BCL1,and there was no significant change in the expression of Gli1.Conclusions: EZH2 may be involved in the activation of Hedgehog signaling pathway in non-small cell lung cancer.The expression of Gli could not be changed by m TOR inhibitor,suggesting that EZH2 activation of Hedgehog signaling pathway is not dependent on m TOR pathway.