Effect Of The HBx Gene Transfection On Proliferation And Invasion Of SUDHL-4 Cell

Background and Objective:Malignant lymphoma(ML) belongs to the tumor lymphatic hematopoietic system.According to the pathological types, it is usually divided into two groups,which are Hodgkin’s lymphomas(HL) and non-Hodgkin’s lymphoma(NHL).Malignant lymphoma accounts for 4% in the total number of emerging malignant tumors every year in China, and it is one of the nine common tumors which came in tenth in cancer deaths. And about 85%~90% of ML is NHL. Diffuse large B cell lymphoma(DLBCL) has high heterogeneity in histological morphology, genetic characteristics, immune phenotype and the clinical manifestations, and it is the most common histologic subtypes in the NHL. In the process of the treatment of patients with lymphoma, we can find more and more hepatitis virus positive patients. The relationship between them gets the attention of various research organizations and relevant researchers. A large number of clinical research data also proves that the rate of NHL patients with hepatitis B virus(HBV) infection is significantly higher than that of the general population and other relevant entities tumor patients, malignant lymphoma with HBV infection rate is next only to cancer of the liver; and the reactivation of HBV in NHL was more marked in the treatment of patients with HBV infection, these studies show that HBV infection has a certain correlation with the incidence of lymphoma.HBV has four ORFs, which are S, C, P and X. HBx is the smallest ORF and encodes 145 amino acids. HBx can induced the formation of hepatocellular carcinoma(HCC) and promote its development through a variety of functions, and inhibite hepatocellular apoptosis occurred which plays a huge role in the process of HCC. But the present study and related reports cannot explain the mechanisms of HBV infection in the occurrence and progress of NHL. Therefore, Lymphoma cells with hepatitis B virus(HBV) model was established through the liposome transfection. Afterwards, the cell lines were chosen as the research object to research the effects HBx on the proliferation of B-NHL and discuss the possible mechanism of the action. In our study, it could provide a new way to screen effective drug and supply more powerful theoretical basis for the treatment of lymphoma with HBV infection.Methods:1. The HBx eukaryon expression vector pcDNA3.1-X was established through recombination DNA technique.2. The HBV X gene eukaryon expression vector pcDNA3.1-X was transiently transfected into SUDHL-4 cell by lipid-media transfection. The reconstituted plasmid pcDNA3.1-X and empty vector pcDNA3.1 were transfected into SUDHL-4 cells which were named SUDHL-4-HBx and SUDHL-4-con. Untransfected SUDHL-4 and SUDHL-4-con were used as controls. The CCK8 experiments were done to detect the reproductive ability of cells in 24 h, 48 h, 72 h and 96 h. Cell apoptosis and cell cycle were detected by flow cytometer.3. The pcDNA3.1-X was transfected into SUDHL-4 cell by lipid-media transfection, and screening the stable transfection cell line SUDHL-HBx by G418.The CCK8 experiments were done to detect the reproductive ability of cells in 96 h.The adhesion abilities of the three kinds cells were tested by modified MTT assay,Transwell Chambers’ s experimental method was used for evaluating the migration and invasion of these three kinds of cells.Results:1. The reconstituted plasmid pcDNA3.1-X was identified by restriction enzymedigestion and nucleotide sequencing.2. HBV x gene m RNA were expressed in the SUDHL-4 cell transfected with the HBx. Compared with SUDHL-4 cell and SUDHL-4-con cell, the cell proliferation slow down and cell cycle in SUDHL-4-HBx cell was obviously changed,accompanied with high rate of apoptosis.3. HBV x gene m RNA and protein was expressed in the stable transfection cell line SUDHL-4-HBx. Compared with SUDHL-4 cell and SUDHL-4-con cell, the proliferative capacity, migration and invasion in SUDHL-4-HBx cell were obviously increased. No significant change in the cell cycle and cell apoptosis rate is significantly reduced.Conclusion:1. The SUDHL-4-HBx cell strain that expressing HBx gene mRNA and the protein has been established successfully.2. Introduction of HBx into SUDHL-4 cells can change the cell cycle and promote cell apoptosis, accompanied with low rate of proliferation.3. The cell proliferation capacity, migration and invasion of the stable transfection cell line SUDHL-4-HBx were obviously increased with the decrease of the cell apoptosis rate.