The Effect And Mechanism Of Transcription Factor EGR1 On The Malignant Biological Behavior Of Pancreatic Cancer

Background:Pancreatic ductal adenocarcinoma(PDAC)with a high degree of malignancy is one of the deadliest cancers in the world.The prognosis of patients with pancreatic cancer remains relatively poor,with a five-year survival rate of about 11%.Since early symptoms are not obvious,local and distant metastasis had occurred when it was found.A small part of patients has the opportunity to accept surgical resection,however they often have distant metastasis within one year,leading to deterioration of the condition.EpithelialMesenchymal Transformation(EMT)refers to the process of epithelial cells transforming into mesenchymal cells,which is one of the pathological mechanisms of cancer progression.EMT promotes the migration,invasion and distant metastasis of pancreatic cancer.The expression and the role of transcription factor EGR1 in pancreatic cancer have not been reported.Objective:(1)To investigate the expression of EGR1 in pancreatic cancer and normal pancreatic tissue;(2)To explore the relation between EGR1 and clinicopathological features and prognosis of patients with pancreatic cancer;(3)To explore the effect of EGR1 on malignant biological behaviors such as migration and invasion of pancreatic cancer cell and the inner mechanism.Methods:(1)The expression of EGR1 in pancreatic cancer tissue and normal pancreatic tissue was compared using TCGA and GTEx databases.The expression of EGR1 between pancreatic cancer and normal pancreatic tissues was compared by immunohistochemistry staining in an 80-patient tissue microarray.The relation of EGR1 and clinical features and prognosis of patients with pancreatic cancer were further explored.(2)qRT-PCR and Western blot were used to investigate the expression of EGR1 mRNA and protein in pancreatic cancer cell lines.SiRNA was used for gene knockdown in pancreatic cancer cells and overexpression plasmid was used to overexpress genes in pancreatic cancer cells.Transwell chamber was applied to estimate the migration and invasion ability of pancreatic cancer cells.SRB methods were used to evaluate the proliferation and chemoresistance ability.(3)The GSEA tool was used to explore the mechanism of EGR1 affecting the progression of pancreatic cancer.Immunofluorescence was used to show and evaluate the expression of E-cadherin in pancreatic cancer cells.The binding of transcription factor EGR1 and SNAI2 gene promoter sequences was verified by ChIP-qPCR experiment.The Dual luciferase reporter gene experiment was conducted to test the activation of SNAI2 gene promoter.P300/CBP inhibitor GNE-272 was used to explore the relationship between EGR1 and transcription co-factor p300/CBP.The Co-IP experiment was used to verify the protein interaction between EGR1 and p300/CBP.Results:(1)EGR1 was highly expressed in pancreatic cancer tissues and lowly expressed in normal pancreatic tissues.High expression of EGR1 was associated with poor prognosis and metastasis of pancreatic cancer.EGR1 was highly expressed in pancreatic cancer cell lines and lowly expressed in normal pancreatic cells.Knockdown of EGR1 significantly inhibited the migration and invasion of pancreatic cancer cells.Knockdown of EGR1 significantly inhibited the proliferation rate of pancreatic cancer cells,while overexpression of EGR1 significantly promoted the proliferation rate of pancreatic cancer cells.Knockdown of EGR1 significantly weakened the chemotherapy drug resistance of pancreatic cancer cells,and the overexpression of EGR1 significantly enhanced the chemotherapy drug resistance of pancreatic cancer cells.(2)GSEA showed that EGR1 was associated with epithelial-mesenchymal transformation and cancer metastasis.Western blot images showed that EGR1 regulated the expression of EMT-related marker.The results of immunofluorescence showed that EGR1 negatively regulated the expression of E-cadherin.RNA correlation analysis showed that EGR1 was positively correlated with the EMT-related transcription factors such as SNAI1,SNAI2,ZEB1,etc.qRT-PCR and ChIP-qPCR confirmed that EGR1 promotes the EMT process of pancreatic cancer through the transcription of SNAI2.Dual luciferase reporter gene assay confirmed that EGR1 bound to SNAI2 promoter sequences.(3)The results of rescue experiment showed that knockdown of EGR1 reduced the migration and invasion ability of pancreatic cancer cells and further overexpression of SNAI2 increased migration and invasion ability of pancreatic cancer cells.Overexpression of EGR1 increased the migration and invasion ability of pancreatic cancer cells and further knockdown of SNAI2 decreased the migration and invasion ability of pancreatic cancer cells.At the same time,immunofluorescence results showed that the expression of SNAI2 counteracted the negative regulation of E-cadherin expression by EGR1.(4)After adding GNE-272,the mRNA and protein levels of SNAI2 were both downregulated,which proved that the expression of SNAI2 was regulated by p300/CBP.When EGR1 was overexpressed and GNE-272 was added,the expression of SNAI2 was increased and subsequently decreased,proving that the transcriptional regulation of EGR1 on SNAI2 was affected by p300/CBP.The Co-IP experiment further proved the combination of EGR1 with p300/CBP.The ChIP-qPCR experiment proved that the transcriptional regulation of EGR1 on the SNAI2 promoter was regulated by p300/CBP.Conclusion:EGR1 is highly expressed in pancreatic cancer and further associated with poor prognosis and lymph node metastasis in pancreatic cancer patients;EGR1 promotes the migration and invasion of pancreatic cancer cells through transcriptional upregulation of SNAI2 expression;P300/CBP plays an auxiliary role in the transcriptional activation of the SNAI2 gene by EGR1.