GOLT1B Activates The EGR1/DKK1/β-catenin Pathway To Promote The Invasion And Metastasis Of Hepatocellular Carcinoma

HCC(hepatocellular carcinoma)is the predominant type of primary liver cancer(PHC),accounting for more than 90% of PHC cases.Liver cancer is a leading cause of cancer-related deaths and ranks sixth in terms of incidence.China has a high incidence rate of HCC,with the number of cases and deaths accounting for over half of the global total.Invasion and metastasis are the primary reasons for HCC recurrence.However,the underlying mechanism of HCC development remains unknown.As a result,finding effective molecular markers for the occurrence and recurrence of hepatocellular carcinoma is essential.Research indicates that Golgi-related genes play a crucial role in the regulation of cancer development.GOLT1 B,a Golgi vesicle transport protein,is a member of the same family as GOLT1 A,which has been shown to regulate tamoxifen sensitivity in breast cancer and promote cell proliferation in lung cancer.However,the role of GOLT1 B in regulating tumor metastasis has not yet been reported.The purpose of this study was to examine the effect of downregulating GOLT1 B on the migration and invasion abilities of liver cancer cells,and to uncover the underlying mechanisms involved.This study utilized Hep G2 and Li-7 liver cancer cell lines in vitro to achieve these goals.The findings of this study contribute to a better understanding of liver cancer treatment and could provide crucial experimental data for future clinical therapies.Objectives:Exploring the effects of downregulation of GOLT1 B on the migration and invasion capabilities of liver cancer cells and associated mechanisms.Methods:1.In the TCGA database,screen for the differential expression of the GOLT1 B gene through bioinformatics methods and perform clinical survival analysis.Use the Kaplan-Meier website to analyze the correlation between GOLT1 B expression and clinical survival in HCC patients.2.Establish GOLT1 B knockdown Hep G2 and Li-7 cell lines,and verify the knockdown efficiency using q-PCR and Western Blot analysis.3.Utilize the scratch and Transwell assays to investigate the effect of GOLT1 B knockdown on the migration and invasion abilities of liver cancer cells.4.Conduct RNA-seq analysis to analyze the differential gene expression of the GOLT1 B knockdown Hep G2 cell line.5.Perform Western Blot analysis to assess the impact of GOLT1 B knockdown on the expression levels of EGR1,DKK1,β-catenin,and β-catenin nuclear protein in liver cancer cells.6.Conduct a chromatin immunoprecipitation(Ch IP)assay to examine the binding of EGR1 and DKK1 to their promoter regions in liver cancer cells.7.Luciferase reporter detects the location of the binding site of the promoter of the EGR1 and DKK1 gene.8.Utilize the scratch and Transwell assays to evaluate the effect of rescue experiments on the migration and invasion of liver cancer cells.9.Perform Western Blot analysis to analyze the expression of EGR1,DKK1,β-catenin,and β-catenin nuclear protein in the rescue experiments.10.Establish a lung metastasis model in nude mice using Hep G2 liver cancer cells,and intravenously inject GOLT1 B knockdown Hep G2 liver cancer cells into the mice.Conduct periodic body weight measurement,excise the lung tissue,and count lung metastatic lesions to investigate the effect of GOLT1 B knockdown on tumor metastasis in vivo.Results:1.In liver cancer patients,the levels of GOLT1 B in liver cancer tissue are significantly elevated,and the expression of GOLT1 B in patients has a negative correlation with overall survival.2.The suppression of GOLT1 B has the ability to reduce the migration and invasion of liver cancer cells.3.The suppression of GOLT1 B leads to a significant increase in the protein expression of EGR1 and a decrease in the expression of DKK1 in liver cancer cells.4.The suppression of GOLT1 B significantly inhibits the nuclear entry of β-catenin in liver cancer cells.5.EGR1 targets the promoter region of the DKK1 gene directly.6.The suppression of GOLT1 B reduces the migratory capacity of liver cancer cells in a mouse model.Conclusions:1.Decreasing the expression of GOLT1 B has the potential to reduce the ability of liver cancer cells to migrate and invade.2.The reduction of GOLT1 B expression may inhibit the invasive and migratory capacity of liver cancer cells via the EGR1/DKK1/β-catenin signaling pathway.