HBx Regulate The DLBCL Function Of Proliferation Through The MEK/ERK-FBW7-c-Myc Axis

Background and purposeLymphoma is the most common malignancy of hematologic malignancies,its morbidity and mortality seriously threaten human survival and health.lymphoma can be divided into Hodgkin’s lymphoma(HL)and non-Hodgkin’s lymphoma(NHL).NHL is the major lymphoma,accounting for 80% to 85%.Diffuse large B cell lymphoma(DLBCL)is the most common of B-cell NHL,accounting for about 50%.DLBCL is highly heterogeneous in etiology,clinical manifestations,treatment and prognosis.Currently,about 60%-70% of patients with DLBCL can be cured after standard clinical treatment,but there are still 30%-40% of patients with refractory and recurrence.Studies have shown that Hepatitis B Virus(HBV)infection is associated with lymphoma,and China is a high prevalence area for HBV infection.In 2022,about 320 million people were infected with HBV worldwide,including nearly 90 million in China.According to data statistics,among all subtypes of NHL,HBV infected patients have the highest risk of developing DLBCL,and HBV-positive DLBCL patients have the characteristics of early onset age,late stage and poor prognosis,while the traditional chemotherapy regimen is not effective,and the survival time is significantly shorter than HBV-negative DLBCL patients.Therefore,it is extremely important to further study the occurrence and development mechanism of HBV-positive DLBCL,find new potential therapeutic targets,and improve the survival rate of patients.However,the current research on the mechanism of correlation is progressing slowly.Over the years,studies on the correlation between HBV infection and DLBCL have focused on the key molecules of HBV serum antigen markers HBs Ag,HBc Ag and HBe Ag,but their expression has not been confirmed in the pathological tissues of HBV-positive DLBCL patients,so it is difficult to construct a dual disease model of HBV infection and DLBCL.Our research group’s previous studies have shown that HBV antigen HBx expression can be detected in the pathological tissues of HBs Ag positive patients with DLBCL,and obtained direct evidence of HBx protein expression in the pathological tissues of HBV-positive patients with DLBCL.HBx expression was found to be associated with high HBV load in HBV-positive DLBCL,Ann Arbor stageⅣ,and poor prognosis,and was positively correlated with c-Myc protein expression.On the basis of previous studies,this project aims to clarify the differential mutant genes of HBV positive patients at the clinical level,explore the influence of HBx expression on the activities of DLBCL cells by constructing human and animal models of HBV antigen HBx protein expression at the in vitro and in vivo levels,and discover the adverse prognostic molecules of HBx expressed DLBCL cells.To provide theoretical basis for the formulation of individual treatment strategies for HBV positive DLBCL patients in clinic.Method1.NGS method was used to detect the gene mutations of FBW7 and c-Myc in paraffin specimens of pathological tissues of 122 patients with DLBCL,among which 51 patients were HBs Ag positive and 71 were HBs Ag negative.2.Lentiviral vector construction human DLBCL cells of stable HBx and carrying Green fluorescent protein(GFP)labeling and purinmycin screening: HBx-SUDHL4(GCB subtype),HBx-U2932(ABC subtype)and control cells: GFP-SUDHL4 and GFP-U2932,and the transfection efficiency was verified by flow cytometry and qRTPCR,and the differential gene FBW7 was further identified by transcriptomic sequencing.3.Molecular expressions of FBW7,c-Myc and related signaling pathways were detected by qRT-PCR and Western-blot.4.Construct HBx-expressed human DLBCL tumor-bearing mouse model to observe the ability of tumor proliferation in vivo;HE staining was used to determine the morphology of tumor tissues and the involvement of organs.The expressions of FBW7 and c-Myc were further detected by immunohistochemical staining,qRT-PCR and Western-blot.5.The proliferation of DLBCL cells was detected by CCK-8 method after transfection of FBW7 with plasmid.Annexin-V PE/7-AAD double staining was used to detect apoptosis of DLBCL.The cell cycle of DLBCL was detected by flow cytometry,and the molecular expression of c-Myc and related signaling pathways was detected by qRT-PCR and Western-blot.Results1.NGS results showed that,compared with HBs Ag positive patients,the differentially mutated genes were mainly FBW7,Myc and TET2.2.Human DLBCL cells with stable expression of HBx,including HBx-SUDHL4 and HBx-U2932,and control cells transfected with empty vector,including GFPSUDHL4 and GFP-U2932,were successfully constructed,and their transfection rates could reach more than 85%.qRT-PCR results showed that HBx m RNA expression levels were significantly increased in HBx-SUDHL4 and HBx-U2932 cells transfected with X genome compared with the negative control group.3.RNA transcriptome sequencing results showed that differentially expressed genes were mainly related to ubiquitin-mediated proteolysis,and FBW7 played an important role in ubiquitin-mediated proteolysis as ubiquitin E3 ligase.4.In vitro results showed that FBW7 expression in HBx-SUDHL4 and HBxU2932 cells was decreased compared with the control group,while c-Myc expression and MEK/ERK phosphorylation levels were significantly increased.5.The results of mouse animal model showed that HBx-U2932 had a short tumor formation time,rapid tumor growth and heavy bone marrow infiltration.Western-blot,qRT-PCR and IHC results showed that compared with the control group,FBW7 expression was decreased in the model group,while c-Myc expression and MEK/ERK phosphorylation level were increased,which was consistent with the results of in vitro experiments.6.The results of DLBCL cells overexpressing FBW7 showed that FBW7 could inhibit the proliferation and promote the apoptosis of HBx-SUDHL4 and HBx-U2932 cells,and could reverse the HBx-induced cell cycle of DLBCL,blocking the cells in the G0/G1 phase.In addition,overexpression of FBW7 also inhibits c-Myc expression and reduces MEK/ERK phosphorylation.Conclusions1.Compared with HBs Ag positive and HBs Ag negative DLBCL patients,the differentially expressed genes were mainly FBW7,Myc and TET2.2.HBx can promote the proliferation of DLBCL cells and inhibit cell apoptosis,suggesting that HBx may be the key molecule driving the poor prognosis of HBVpositive DLBCL.3.HBx can regulate a series of biological events of DLBCL through the MEK/ERK-FBW7-c-Myc axis.4.FBW7 is likely to play a role as a tumor suppressor in HBV-positive DLBCL and is a potential clinical therapeutic target.