HBx蛋白羧基端缺失对肝癌细胞生物学行为的影响

Biological Effects of COOH-terminal Amino Acid Deletions of Hepatitis B Virus X Protein on Hepatocellular Carcinoma Cell Line Huh7

背景与目的:肝癌组织内普遍存在着截短表达的HBx蛋白,这种蛋白可能与肝癌的发生有关,本研究探讨截短表达的HBx蛋白和野生型HBx对人肝癌细胞Huh7生物学行为的影响。方法:脂质体法介导HBx突变体和野生型HBx重组体转染HBV(-)的Huh7细胞。PCR方法扩增Neo基因检测质粒DNA片段插入。通过MTT、平板克隆形成实验、流式细胞仪和裸鼠成瘤实验检测稳定转染细胞的生物学活性。结果:HBx3′-20和HBx3′-40组细胞生长速度较HBx3′-30组明显增快(P<0.05);HBx3′-20组[(17.34±2.77)%]和HBx3′-40组[(18.36±2.61)%]克隆形成率明显较HBx3′-30组[(7.31±1.44)%]和pcDNA3组[(6.87±2.38)%]高(P<0.05)。细胞周期检测结果显示,对比于野生型HBx组,HBx3′-20组和HBx3′-40组蛋白的表达能加速Huh7细胞由G0/G1期到S期的进程[S期:(36.96±1.82)%vs.(46.20±3.23)%,(53.99±4.02)%,P<0.05],相反,HBx3′-10和HBx3′-30组则出现G1期阻滞,而HBx组与pcDNA3空载体组间[(38.60±1.15)%]在S期无明显变化。裸鼠成瘤实验显示,HBx3′-40组[(3.19±0.34)cm3]成瘤体积明显大于HBx3′-30组[(1.58±0.27)cm3]、HBx组[(1.75±0.15)cm3]和pcDNA3组[(1.67±0.12)cm3],各组瘤重存在显著性差异(P<0.01)。结论:对比野生型HBx组,HBx3′-20和HBx3′-40对Huh7细胞的生长具有明显促增殖作用,HBx3′-30组显示出抑制作用。HBx通过缺失突变修饰其生物学功能,在原发性肝癌发生、发展过程中起着重要作用。

BACKGROUND ＆ OBJECTIVE： The COOH-terminal amino acid deletions of hepatitis B virus X protein （HBx） commonly exist in human hepatocellular carcinoma （HCC）. This study was designed to explore the biological effects of truncated HBx and wild type HBx on HCC cell line Huh7. METHODS= The mutants of HBx with 10, 20, 30, or 40 amino acids deletion at COOH-terminal （HBx3＇-10, -20, -30, -40）, or 37 amino acids deletion at the middle （HBx-XMR） were constructed. The recombinant truncated HBx and wild type HBx expression vectors were transfected into Huh7 cells. The integration of the exogenous vector DNA was detected by Neo gene polymerase chain reaction （PCR）, The biological characteristics of positive clones were analyzed by MTT assay, colony formation assay, flow cytometry （FCM）, and xenograft in nude mice. The expression of HBx3＇-10, HBx3＇-20, HBx3＇-30, HBx3＇-40, HBx-XMR and HBx was detected by immunohistochemi- stry, RESULTS= Huh7 cells grew faster in HBx3＇-20 and HBx3＇-40 groups than in HBx3＇-30 group （P〈 0.05）. The colony formation rate was significantly higher in HBx3＇-20 and HBx3＇-40 groups than in HBx3＇-30 and pcDNA3 groups [（17.34±2.77）% and （18.36±2.61）% vs. （7.31±1.44）% and （6.87±2.38）%, P 〈 0.05]. Compared with wild type HBx, HBx3＇-20 and HBx3＇-40 promoted more cells from G1 phase into S phase in cell cycle [（36.96±1.82）% vs. （46.20±3,23）% and （53.99±4.02）% in S phase, P〈0.05], while HBx3＇-10 and HBx3＇-30 blocked the procedure in G1 phase [（32.30±4.32）% and （30.34±1.47）% in S phase]; no obvious change was found between wide type HBx group and pcDNA3 group [（38.60±1.15）% in S phase]. The volume of xenograft tumor in nude mice was obviously larger in HBx3＇-40 group than in HBx3＇-30, wild type HBx, and pcDNA3 groups [（3.19±0.34） cm^3 vs. （1.58±0.27） cm^3, （1.75±0.15） cm^3, and （1.67±0.12） cm^3]; the tumor weight showed the same trend among the groups. CONCLUSIONS. Compare with wide type HBx, HBx3＇-20 and HBx3＇-40 could promote the proliferation of Huh7 cells, but HBx3＇-30 has the contrary effect. HBx mutants might play a role in the development of HCC through modifying the biological functions of HBx.