Effect of combo mutation of core promoter on expression of proliferation cell nuclear antigen, nuclear factor-κB and p21

Objective The A1762T/G1764A/T1753A/T1768A combo mutant core promoter was constructed through the gene synthesis,and infected into L-02 cells.The expression of proliferation cell nuclear antigen (Ki-67),nuclear factor-κB (NF-κB) and p21 was investigated.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 293T cells for virus packaging through calcium turn method,and then infected into L-02.The expression of HBXFlag,Ki-67,NF-κB and p21 was detected by Western blotting.Results The pLenO-RTP-CM-HBX core promoter lentivirus carrier could infected into L-02 cells with high efficiency (＞ 95％).The HBX-Flag could be 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 Ki-67,NF-κB,p21 expression gray in mutant group were 2.103 ±0.346,2.201 ±0.486,0.771 ±0.338; the expressions gray in light group were 0.221 ± 0.016,0.232 ± 0.117,2.032 ± 0.215 ; and the expressions gray of Ki-67,NF-κB,p21 in blank control group were 0.323 ±0.017,0.334 ±0.207,2.141 ±0.305.The expression of Ki-67 and NF-κB in mutant group was increased,and that of p21 was reduced compared to light group and the blank control group (P ＜ 0.05).There was no significant difference between the light group and the blank control group.Conclusion The combo mutation of core promoter can influence the expression of Ki-67,NF-κB and p21. Key words: Hepatitis B virus;  Core promoter;  Combo mutation