[Biological effects of COOH-terminal amino acid deletions of hepatitis B virus X protein on hepatocellular carcinoma cell line Huh7].
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 immunohistochemistry.


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 G(1) 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 G(1) 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) cm3 vs. (1.58+/-0.27) cm(3), (1.75+/-0.15) cm3, and (1.67+/-0.12) cm3]; 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.
Liu
Xiao-Hong
XH
Department of Pathology, The Second Military Medical University, Shanghai, P. R. China.
Zhu
Ming-Hua
MH
Cao
Xiao-Zhe
XZ
Zheng
Jian-Ming
JM
Chen
Ying
Y
D000595:Amino Acid Sequence; D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000473:pathology* / Q000821:virology; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D005822:Genetic Vectors; D006515:Hepatitis B virus / Q000235:genetics; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology* / Q000821:virology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D010957:Plasmids; D011897:Random Allocation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D054334:Viral Regulatory and Accessory Proteins
D011994:Recombinant Proteins; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein