The Mechanism Of GLI2/DEC1/ZEB1 Pathway Regulating EMT And Cisplatin Sensitivity Of Gastric Cancer Cells

BackgroundGastric cancer is a malignant tumor with high morbidity and mortality in the world.It has the characteristics of insidious onset,easy metastasis and recurrence,and poor prognosis.Recent studies have found that Epithelial-Mesenchymal transition(EMT)of tumor cells may be a key reason for tumor metastasis and treatment resistance.EMT is a process in which epithelial cells lose polarity and adhesion,and transform into mesenchymal cells with migration and invasion ability.After the EMT of tumor cells,the migration and invasion ability of cells is enhanced,and the chemotherapy resistance appears.Hedgehog signaling pathway is the core pathway for tumor targeted therapy and improvement of chemotherapy resistance.At present,the research on Hedgehog signaling pathway mainly focuses on the classical SMO ligand-activated Hedgehog signaling pathway.SMO ligand inhibitors have been used in clinical treatment of hematological diseases and basal cell carcinoma.However,the results of clinical trials have shown that its therapeutic effect on gastric cancer is not ideal,suggesting that the non-canonical Hedgehog signaling pathway may play a more important role in gastric cancer.The transcription factor Glioma-associated oncogene 2(GLI2)is the core transcription factor in the non-canonical Hedgehog signaling pathway,but its role in the occurrence and development of gastric cancer,especially the regulation of EMT,is still unclear.Based on the previous research work,we clarify the regulatory mechanism of GLI2 on EMT and chemotherapy resistance of gastric cancer cells from the perspective of transcriptional regulation,which provides a theoretical basis for understanding the metastasis and treatment resistance of gastric cancer,and provides new ideas for targeted therapy of gastric cancer.Objective1.Investigate whether GLI2 plays an important role in the occurrence and development of gastric cancer by regulating EMT and cisplatin sensitivity.2.Investigate the transcriptional regulation of GLI2 on the downstream molecule Differentiated embryo-chondrocyte expressed gene 1(DEC1).3.Clarify the role of GLI2/DEC1/ZEB1 pathway in EMT and cisplatin resistance of gastric cancer cells.Methods1.TCGA and GEO databases were used to analyze the expression of GLI2 in gastric cancer tissues and different molecular subtypes;Kaplan-Meier plotter was used to analyze the correlation between GLI2 expression and the prognosis of gastric cancer.2.GLI2 and DEC1 stable expression/knockdown cell lines were constructed by lentivirus transfection in gastric cancer cells,and verified by PCR and Western blot.3.EdU,Transwell,and scratch assays were used to verify the effects of GLI2,DEC1,and ZEB1 on the proliferation and migration of gastric cancer cells.Western blot was used to verify the regulation of GLI2,DEC1 and ZEB1 on EMT-related molecules.4.CCK-8 assay was used to detect the IC50 of cisplatin,and Transwell assay was used to detect the migration ability of cells.The changes of EMT and cisplatin sensitivity were observed.Western blot was used to explore the effect of cisplatin on the expression of EMT and proliferation-related molecules in gastric cancer cells.5.The potential binding sites of DEC 1 and ZEB1 promoter regions were analyzed by online websites.PCR,Western blot and ChIP experiments were used to verify the transcriptional regulation relationship between GLI2-DEC1 and DEC1-ZEB1.6.The expression of GLI2,DEC1 and ZEB1 in gastric cancer and its relationship with clinicopathological parameters of gastric cancer patients were analyzed by immunohistochemical staining(IHC).The correlation between the expression of GLI2,DEC1 and ZEB1 was analyzed by correlation test.To investigate the effect of combined expression of GLI2 and DEC1 on the overall survival of gastric cancer patients.7.Subcutaneous tumor formation and tail vein metastasis experiments were performed in nude mice,and combined with cisplatin treatment,to explore the effect of GLI2 and DEC1 expression changes on the growth and metastasis of gastric cancer cells in vivo,as well as the effect of cisplatin treatment.8.Investigate the effect of GLI2 and DEC1 on the expression of related molecules in nude mice tumors by IHC.Results1.Bioinformatics analysis showed that GLI2 was highly expressed in gastric cancer tissues,which was associated with poor prognosis and was highly expressed in EMT and mesenchymal phenotype.2.IHC results showed that GL12 expression was increased in gastric cancer tissues compared with adjacent normal tissues;The expression of GLI2 is significantly correlated with the differentiation degree,pathological grade,lymph node metastasis and prognosis of gastric cancer patients.3.GLI2 co-regulated EMT and cisplatin sensitivity of GC cells by activating DEC1 expression in vitro.4.In vivo study demonstrated that GLI2 and DEC1 combined regulate the growth and metastasis of gastric cancer cells and the therapeutic effect of cisplatin.5.ChIP assay confirmed that GLI2 transcriptionally regulated DEC1 expression.6.Western blot and functional assays demonstrated that DEC1 regulated the expression of ZEB1 and affected the EMT process and cisplatin sensitivity of gastric cancer cells.ChIP assay showed that DEC1 transcriptionally regulated ZEB1 expression by binding to the ZEB1 promoter region.7.IHC of clinical patient tissues showed that the expression levels of DEC 1 and ZEB1 were increased in gastric cancer tissues and were significantly correlated with the prognosis.The expressions of GLI2,DEC1 and ZEB1 were highly positively correlated in gastric cancer tissues.Conclusions1.GLI2 promotes the malignant progression of gastric cancer by promoting EMT and cisplatin resistance of gastric cancer cells.2.DEC 1 promotes the expression of ZEB1 by binding to the promoter region of ZEB1,and ultimately promotes EMT and cisplatin resistance of gastric cancer cells.