Mechanism Of HBx Regulating The Proliferation,Migration And Invasion Of Hepatocellular Carcinoma Cell Through MiR-187-5p/CDH2

Objective: Hepatitis B virus x protein HBx(Hepatitis B virus x protein,HBx)is an important non-structural and multifunctional regulatory protein of hepatitis B virus(HBV).It is also one of the main factors that induce HBV-related Primary Hepatocellular Carcinoma.The transcription factor E2F1 is the target of a variety of oncogenic viral proteins and plays an important role in regulating the cell cycle.The transcription factor FOXP3,a key factor regulating the expression of immunosuppressive molecules,is involved in the proliferation,migration and invasion of various tumors.Studies have found that HBx can regulate non-protein-coding miRNAs,but the specific mechanism is not yet clear.In this study,we explored the interaction mechanism between HBx;miR-187-5p and transcription factors E2F1 and FOXP3 in hepatocellular carcinoma cells,in which the HBx/E2F1/FOXP3/miR-187-5p/CDH2 signaling pathway plays a role in the proliferation,migration,and invasion of hepatocellular carcinoma cells.The role and mechanism of HBx,migration and invasion,aiming to further clarify the molecular pathogenic mechanism of HBx in HCC(Hepatocellular carcinoma,HCC),and provide new ideas for the clinical diagnosis and treatment of HCC.Methods: The miRNAs of human normal liver cells LO2,hepatocellular carcinoma cells Huh-7 and Hep G2 cells were extracted,and the expression difference of miR-187-5p was detected by qRT-PCR experiment.Different doses of pc DNA3.1-HBx expression plasmids were transfected into Huh-7 cells in logarithmic growth phase,and the expression changes of miR-187-5p in hepatocellular carcinoma cells after overexpression of HBx were detected.The miR-187-5p mimics,inhibitors and their respective controls were transfected into Huh-7 cells,and the CCK-8 experiment,Ed U experiment,scratch experiment and Transwell experiment were performed to verify that miR-187-5p has effects on hepatocellular carcinoma cells Huh-7 The effects of proliferation,migration and invasion.Construct wild-type and mutant luciferase reporter gene plasmids with FOXP3 binding site on miR-187-5p promoter,and use luciferase reporter experiment to understand the binding of FOXP3 and miR-187-5p promoter;Western Blotting detects miR-187-5p expression of downstream target gene CDH2.Construct wild-type and mutant luciferase reporter gene plasmids with the E2F1 binding site on the FOXP3 promoter to understand whether the E2F1 and FOXP3 promoters are combined.The Co IP experiment verifies whether HBx and E2F1 interact.Construct a cell line stably expressing miR-187-5p in hepatocellular carcinoma cell Huh-7,establish an in vivo mouse hepatocellular carcinoma xenograft model,observe the effect of miR-187-5p overexpression in vivo on tumor growth;collect xenograft tissue samples and freeze Sections,immunohistochemistry and Western Blotting to detect the expression of Ki67 and CDH2 proteinResults: Through q RT-PCR experiments,we found that HBx inhibited the expression of miR-187-5p in hepatocellular carcinoma cells.After transfection of miR-187-5p mimic and its control in Huh-7 cell,miR-187-5p inhibited the proliferation of hepatocellular carcinoma cells by the Ed U experiment and CCK-8 experiment.The scratch experiment and Transwell experiment results indicated miR-187-5p inhibited the migration and invasion of hepatocellular carcinoma cells.Luciferase experiment results showed that miR-187-5p inhibited the expression of CDH2 protein by targeting 3’-UTR.The results of the Co IP experiment and Western Blotting showed that HBx interacted directly with E2F1 to promote the expression of FOXP3.The results of the Luciferase experiment showed that FOXP3 inhibited the expression of miR-187-5p by binding to the promoter.Overall the HBx/E2F1/FOXP3/miR-187-5p/CDH2 signaling pathway is formed to promote the proliferation,migration,and invasion of hepatocellular carcinoma cells.Conclusion: This study explored a new molecular mechanism by which HBx affects the occurrence and development of HBV-related HCC through miR-187-5p/CDH2.HBx promotes the expression of FOXP3 by interacting with E2F1,thereby inhibiting miR-187-5p,leading to up-regulation of CDH2 expression,forming a complete HBx/E2F1/FOXP3/miR-187-5p/CDH2 signaling pathway,and promoting the proliferation of hepatocellular carcinoma cells,Migration and invasion are expected to provide new targets for the clinical diagnosis of HBV-related primary hepatocellular carcinoma.