HBx Inhibits GATA4 Expression And Its Transferring Into Nucleus Via Activated Wnt/beta-catenin Pathway In Hepatocellular Carcinoma Cell Line HLE

Objective: After hepatitis B virus(HBV)infects liver cells,the X protein(HBx)encoded by the X gene in the viral genome accumulates in the cytoplasm of hepatocytes and is closely related to the malignant transformation of hepatocytes.The transcriptional regulator GATA4 is synthesized in the cytoplasm and transferred into the nucleus.The function of GATA4 protein is regulation cell reprogramming and differentiation.Its function inactivation promotes the occurrence of liver cancer.The wnt/beta-catenin pathway is a nuclear transduction pathway that accelerates the progression of liver cancer cells after activation.Previous studies have found that in liver cancer cells expressing GATA4,HBx protein accumulated due to HBV infection can regulate the process of GATA4 transferring into the nucleus from cytoplasm.The whole process is accompanied by high expression of wnt/beta-catenin.It is currently unknown whether HBx protein regulated GATA4 transferring into the nucleus via wnt/beta-catenin.So we hypothesized that HBx protein might activate the wnt/beta-catenin pathway to regulate GATA4 transferring into the nucleus,subsequently promoting liver cancer progression.Methods:(1)Construction of a lentivirus-mediated overexpression vector CMV-HBx.(2)Detection of m RNA levels of HBx,wnt/beta-catenin and GATA4 by RT-PCR.(3)Western blot analysis of protein levels of HBx,wnt/beta-catenin and GATA4.(4)The expression positions of HBx,wnt/beta-catenin and GATA4 was detected by immunofluorescence.(5)Beta-catenin activity was specifically inhibited by the addition of the beta-catenin antagonist PRI-724.Results: Successful construction of HBx stable cell expression strain HLE-HBx mediated by lentiviral expression vector CMV-HBx.The results of RT-PCR and western blot showed that HBx was successfully expressed in HLE-HBx cells.In addition,the expression of wnt/beta-catenin was increased,and the total protein levels of GATA4 was decreased in HLE-HBx cells compared to the control.Laser confocal observation showed that in HLE overexpressing HBx,the process of GATA4 transferring into the nucleus from cytoplasm is decreased.CO-IP experiments suggestde that GATA4 may interact with beta-catenin protein in cytoplasm.Conclusion:HBx expressed in human hepatoma cell line HLE inhibits the expression of total GATA4 and its transferring into nucleus by activating the wnt/beta-catenin signaling pathway.The activated beta-catenin binds to GATA4,inhibits its transferring into nucleus,and leads to the functional inactivation of GATA4,thereby promoting malignant transformation of liver cells.