SMAD2 Transcriptional Activation Of BUB1 Enhances TGFβ Signaling Pathway And Promotes DNA Damage,Proliferation And Migration In Liver Cancer

Background:Hepatocellular carcinoma(HCC)is the seventh most common malignancy worldwide and the second leading cause of cancer-related mortality.Therefore,it poses a significant threat to human health.About 780,000 people are diagnosed with HCC each year,and a large proportion of them are diagnosed with advanced tumors.In recent years,significant progress has been made in the treatment of HCC,but the survival rate of these patients is still less than 20%,and the prognosis is poor.Therefore,exploring the development mechanism of HCC cells at the molecular level is expected to reveal new therapeutic targets and develop more effective therapeutic strategies.In this study,the TCGA and ICGC databases were used to analyze the expression of Budding Uninhibited by Benzimidazole 1(BUB1)in HCC,and the relationship between the expression level of BUB1 in HCC samples and the clinicopathological data and prognosis of patients was studied.The influence of BUB1 on the biological behavior of HCC cells was studied through in vitro and in vivo experiments,aiming to reveal the relevant mechanism of the clinical significance and prognostic effect of BUB1 in HCC,and provide new targets for the molecular diagnosis and targeted therapy of HCC.Methods:(1)Differential expression genes in HCC were analyzed using the TCGA and ICGC databases,and significantly upregulated genes were selected for correlation analysis.(2)The expression of BUB1 in HCC tissues was detected by immunohistochemistry,and its relationship with clinical pathological parameters and prognosis was studied.The effect of BUB1 on HCC cell proliferation was measured using CCK-8,colony formation,and EDU assays.The effect of BUB1 on HCC cell migration was evaluated using Transwell and scratch assays.The effect of BUB1 on tumor growth in vivo was observed using a subcutaneous tumor model.The effect of BUB1 on the localization and expression of γh2AX in HCC cells was evaluated using immunofluorescence staining.The effect of BUB1 on γh2AX expression and TGFβsignaling transduction were analyzed using Western blot.(3)Transcription factors that may regulate BUB1 expression were searched using bioinformatics methods,and the binding site of SMAD family member 2(SMAD2)in the BUB1 promoter region was explored.The expression of SMAD2 in HCC and its correlation with prognosis were analyzed using databases and immunohistochemistry.The correlation between SMAD2 and BUB1 in HCC tissues was demonstrated using bioinformatics.The localization and expression of SMAD2 and BUB1 in HUH7 cells were analyzed using immunofluorescence staining.The regulatory effect of SMAD2 on BUB1 was analyzed using q RT-PCR and Western blot.The positive transcriptional regulatory effect of SMAD2 on BUB1 was confirmed using a dual luciferase reporter gene experiment.(4)Functional rescue experiments(CCK-8,colony formation,EDU,scratch healing,immunofluorescence staining and Western blot)were conducted to demonstrate that SMAD2 promotes HCC cell TGFβ signaling,DNA damage,proliferation,and migration in a BUB1-dependent manner.(5)The effect of the BUB1 inhibitor 2OH-BNPP1 on HCC cell proliferation was measured using CCK-8 and EDU assays.The effect of BUB1 on HCC cell migration was evaluated using scratch healing and Transwell assays.The effect of the BUB1 inhibitor 2OH-BNPP1 on γh2AX expression and TGFβ signaling in HCC cells were evaluated using immunofluorescence staining and Western blot.The effect of 2OHBNPP1 on tumor growth in vivo was observed by constructing a subcutaneous tumorigenic model.Results(1)BUB1 is highly expressed in HCC and is associated with poor prognosis in patients.The vitro cell experiments show that BUB1 enhances the proliferation and migration ability of HCC cells.The vivo experiments demonstrate that BUB1 promotes tumor growth.BUB1 regulates TGFβ signaling and affects the degree of DNA damage.(2)The transcription factor SMAD2 has a binding site with the BUB1 promoter region;SMAD2 positively regulates the expression of BUB1 in HCC and promotes the upregulation of BUB1 through transcriptional activation.(3)Through Rescue experiments,SMAD2 transcriptionally activates BUB1 to enhance the TGFβ signaling pathway,promoting DNA damage,proliferation,and migration in liver cancer.(4)The BUB1 inhibitor 2OH-BNPP1 inhibits the activation of TGFβ signaling,DNA damage,proliferation,and migration of HCC cells.The vivo experiments show that 2OH-BNPP1 inhibits the growth of subcutaneous tumors.Conclusion(1)BUB1 is an adverse prognostic factor for patients with HCC,and its presence independently increases the patient’s risk.(2)SMAD2 can bind to the BUB1 promoter,thereby promoting BUB1 expression in HCC.(3)In addition,SMAD2 transcriptionally activates BUB1 to enhance the TGFβsignaling pathway,promoting DNA damage,proliferation,and migration in HCC.(4)BUB1 inhibitor 2OH-BNPP1 inhibits TGFβ signaling,DNA damage,proliferation,and migration in HCC cells.Therefore,BUB1 may be a potential target for treating HCC.