HRG Switches HCC Cell Fate From TNFR1-Mediated Cell Survival To Cell Death

Background and ObjectiveHepatocellular carcinoma(HCC)is the sixth most common cancer,and the fourth leading cause of cancer-related death worldwide.HCC is a serious threat to the health of Chinese people.Tumor necrosis factor receptor 1(TNFR1)is a ubiquitous membrane receptor on the surface of various types of cells.TNFR1 is activated by TNF-α and induces cell apoptosis.Histidine-rich glycoprotein(HRG)is a multi-domain plasma glycoprotein,which can inhibit abnormal tumor blood vessels and tumor growth.The role of HRG in HCC and its related mechanisms have not been reported.Therefore,this study intends to explore the role of HRG in the occurrence and development of HCC and the related molecular mechanism.MethodsIn the study,first by constructing two TNFR1 complex models and screened HRG through immunoprecipitation and mass spectrometry,the relationship between HRG and TNFR1 was explored through correlation analysis of TCGA data sets and coimmunoprecipitation experiments.The regulation effect of HRG on TNFR1 was determined by western blot and immunoprecipitation.The relationship between HRG and NF-κB was confirmed by immunofluorescence,double luciferase reporter assay and qRT-PCR.The effect of HRG on the proliferation of HCC cells was detected by EdU and subcutaneous tumorigenesis in nude mice.The expression of HRG in liver cancer tissues and its relationship with the prognosis of HCC patients were determined by the analysis of TCGA,GEO data set,qRT-PCR and western blot.Results1.In this study,HRG was selected by immunoprecipitation and mass spectrometry,and the combination of HRG and TNFR1 was predicted by Biogrid website.TCGA correlation analysis indicated that HRG was highly correlated with TNFR1,and the coimmunoprecipitation experiment proved that HRG was combined with TNFR1.2.EdU assay proved that HRG inhibits the proliferation of HCC cells.TUNEL assay proveed that HRG promotes HCC cells apoptosis.Flow cytometry showed that HRG enhanced the sensitivity of tumor cells to 5-fluorouracil.Subcutaneous tumorigenesis in nude mice,HE staining and immunohistochemical detection of ki-67 proved that HRG inhibited the proliferation of liver cancer in vivo.3.The expression,half-life and ubiquitination level of TNFR1 was detected in overexpressed HRG liver cancer cell lines,it was proved that HRG can reduce the ubiquitination degradation of TNFR1 and stabilize its expression in cells.The markers of complex Ⅱ were detected by immunoprecipitation experiment,the data showed that HRG can promote TNFR1 formation complex Ⅱ mediated apoptosis.4.The expression of phosphorylated P65 was detected by immunofluorescence assay in overloaded HRG and HRG knockout cells,it was proved that HRG significantly decreased NF-κB activity.Double-luciferase reporter assay,qRT-PCR for mRNA expression of NF-B related genes,and western blot for phosphorylated P65 in overloaded HRG TNFR1 knockout cells demonstrated that HRG inhibited NF-kB activation and was dependent on TNFR1.qRT-PCR was used to detect HRG mRNA expression in TNF-α and GW4064 treated cells.It was proved that NF-κB could inhibit HRG expression in liver cancer cells.A negative feedback loop exists between HRG-TNFR1-NF-κB.5.Through qRT-PCR and western blot experiments,combined with the analysis of TCGA and GEO data sets,it was proved that low HRG expression in liver cancer tissues was correlated with poor prognosis of liver cancer patients.Conclusion1.HRG combines with TNFR1.2.HRG inhibits the proliferation,promotes apoptosis.3.HRG stabilizes the expression of TNFR1 by reducing its ubiquitination and promote the formation of TNFR1 complex Ⅱ to mediate apoptosis.4.A negative feedback loop exists between HRG-TNFR1-NF-κB.5.Low HRG expression was associated with poor prognosis in HCC patients.