Transfection of L-02 with combo mutant core promoter and its effect on cell proliferation ability

Objective The core promoter (CP) region overlaps with the hepatitis B virus (HBV) X gene.Clinical research has shown that A1762T/G1764A (TA)/T1753A/T1768A mutation in overlapping sequence can increase risk of hepatocellular carcinoma (HCC).In order to establish a kind of cell model of hepatocarcinogenesis,we construct A1762T/G1764A/T1753A/T1768A combo mutant core promoter through the gene synthesis,and then infect the cell L-02.Methods In order to obtain recombinant plasmid pLenO-RTP-CM-HBX,the pLenO-RTP carrier and pUC57-CM-HBX were connected directly after double restriction enzyme digestion.The recombinant plasmid was transfected into 293 T cells for virus packaging through Calcium turn method,and then L-02 cells were infected.The change of proliferation ability after transfection was detected through methyl thiazol tetrazolium (MTT) method.HBX-Flag,S-phase kinase-associated protein-2 (Skp2),proliferating cell nuclear antigen (PCNA),p21 and p53 expression changes were detected by using Western blotting.Results The pLenO-RTP-CM-HBX core promoter lentivirus carrier could infect L-02 cells with high efficiency (＞ 95％),and then HBX-Flag was transcribed,translated,synthetized and secreted in L-02 cells after transfection.The red fluorescence intensity did not change obviously with the extension of incubation time.The proliferation ability,and Skp2 and PCNA expression of cells in combo mutant group was increased,and p21 and p53 expression was reduced compared to light group and blank control group (P ＜ 0.05).There was no significant difference between light group and blank control group.Conclusion The pLenO-RTP-CM-HBX core promoter lentivirus carrier was transfected into L-02 cells successfully,and the L-02 cells after transfection had tendency to become cancerous. Key words: Core promoter;  Hepatitis B virus;  Lentivirus carrier;  Combo mutation