[Polymorphism of hepatitis B virus x gene in hepatocellular carcinoma patients and the biological effects of HBx gene with deletion varations on QSG7701 cells].
OBJECTIVE
To investigate whether there were particular HBx gene mutations associated with hepatocellular carcinoma (HCC) development in patients.


METHODS
The HBx genes were examined in 51 paraffin-embedded tumor tissue samples from patients with HCC and 25 serum samples from HBV carriers from southern China by nested polymerase chain reaction (PCR), single-stranded conformational polymorphism analysis, heteroduplex analysis and DNA sequencing. The HBx genes with deletion variations (HBx-d382, HBx-d431) from tumor tissues were cloned and transfected into QSG7701 cells. Then, the biological characteristics of the transfected cells were analyzed in nude mice by MTT assay, soft agar colony formation assay, flow cytometry and xenografting.


RESULTS
Deletion mutation and point mutation were found in the HBx genes of HCC tumor tissues, and there were some differences between the HBx gene mutations in genotype B and those in genotype C. More mutations were found in genotype C than those in genotype B (t=-2.522, P < 0.05), but the deletion variations (HBx-d382, HBx-d431) were detected in genotype B HBV from HCC liver tissues. The HBx genes with deletion variations (HBx-d382, HBx-d431) were recombinant with pcDNA3 and transfected into QSG7701 cell lines successfully, which established four permanent transfected QSG7701 cell lines, including pcDNA3/HBx-d382/QSG7701, pcDNA3/HBx-d431/QSG7701, pcDNA3/HBx/QSG7701, and pcDNA3/QSG7701. pcDNA3/HBx-d382/QSG7701 and pcDNA3/HBx-d431/QSG7701 grew faster and had more potential colony formative activity than those of pcDNA3/QSG7701. Moreover, pcDNA3/HBx-d382/QSG7701 and pcDNA3/HBx-431/QSG7701 cells inoculated in nude mice produced tumors more rapidly than those of pcDNA3/HBx/QSG7701, and pcDNA3/QSG7701. The volumes of the tumors in nude mice were also obviously larger in pcDNA3/HBx-d382 and pcDNA3/HBx-d431 groups than those in pcDNA3/HBx/QSG7701 and pcDNA3/QSG7701 groups.


CONCLUSIONS
Our results suggest that HBx gene mutations occur frequently in HCC tissues, and the deletion at nt382-400 of the HBx gene might play a role in carcinogenesis of HCC in southern China.
Zhu
Ping-an
PA
Department of Infectious Diseases, Xiangya Hospital, Central South University, Changsha 410008, China.
Tan
De-ming
DM
Peng
Zhong-tian
ZT
Liu
Fei
F
Song
Lin
L
D000328:Adult; D000368:Aged; D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000821:virology*; D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000235:genetics; D005260:Female; D006514:Hepatitis B Surface Antigens / Q000097:blood; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008113:Liver Neoplasms / Q000821:virology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008875:Middle Aged; D017354:Point Mutation; D018807:Polymorphism, Single-Stranded Conformational; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D015534:Trans-Activators / Q000235:genetics*; D014162:Transfection; D054334:Viral Regulatory and Accessory Proteins
D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein