Abstract
AIM To observe the roles of N-ras gene mutation and hepatitis B virus (HBV) infection in the carcinogenesis of hepatocellular carcinoma (HCC) in Guangxi, China.
METHODS The polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and immunohistochemistry were used to detect N-ras gene mutation and HBV infection in 29 cases of HCC.
RESULTS The aberration rates at codon 2-37 of N-ras were 79.31% in HCCs and 80.77% in adjacent non-tumorous liver tissues. More than 2 point mutations of N-ras gene were observed in 22 (75.86%) cases. HBsAg and HBxAg positive rates were 86.2% and 79.3%. There was a parallel tendency between HBV marker detections and the mutation rate of N-ras gene.
CONCLUSION HBV infection and N-ras gene mutation may be involved in the carcinogenesis and development of HCC in Guangxi. Since the aflatoxin B1 contamination is one of risk factors for HCC in this area, it may contribute to the mutation of N-ras gene in carcinogenesis of HCC.INTRODUCTION
Hepatocellular carcinoma (HCC) is one of common malignant tumors in People′s Republic of China. Guangxi is a high incidence area of HCC. Many factors are involved in hepatocarcinogenesis. Many studies revealed that hepatitis B virus (HBV) infection might be a risk factor for hepatocellular carcinogenesis. One theory for hepatocarcinogenesis is that the oncogene(s) may be transactivated by hepatitis B x antigen (HBxAg)^[1] . It is found recently that activation of N-ras gene may be the molecular basis for the carcinogenesis and development of HCC^[2,3] . There have been reports about overexpression of N-ras oncogene in human HCC^[4] , but a few dealt with the roles of N-ras gene mutation and HBV infection, and their relationship with HCC. We analyzed the N-ras gene mutation and HBV infection in HCC by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and immunohistochemistry in 29 cases of human HCC.

MATERIALS AND METHODS
Clinical samples
Surgically resected specimens of HCC (29 cases) were collected in Guangxi Cancer Institute during the period of 1987-1992. Twenty-eight cases of them contained HCC adjacent liver tissues. All samples were fixed with 10% formalin, embedded in paraffin and stained with haematoxylin and eosin (HE).

Immunohistochemistry
Immunostaining was performed by a streptavidin-biotin immunoperoxidase method. Hepatitis B surface antigen (HBsAg) and x antigen (HBxAg) were detected with monoclonal anti-HBsAg antibody, anti-HBxAg antibody and Strept ABC kit (DAKO A/S Denmark).

DNA extraction
Genomic DNA was prepared by the proteinase K-Phenol-Chloroform extraction method.

PCR-SSCP
Oligomers that flank codon 2-37 of N-ras genes were synthesized as primers by the Department of Molecular Medicine, King′s College Hospital, UK. One of them was 5′-end labeled with r-³² P ATP by T4 polynucleotide kinase reaction. The primer sets were as follows:
5′-GACTGAGTACAAACTGGTGG-3′
5′-GGGCCTCACCTCTATGGTG-3′
The amplified product obtained by the PCR was 118bp. DNA 0.1μg extracted from tissues was added into 9μl PCR mixture (containing 1pmol/L primer, 0.2mmol/L dATP, dGTP, dCTP, dTTP, 0.25U Taq DNA polymerase, 50mmol/L KCl, 10mmol/L Tris, 2.5mmol/L MgCl₂ and 0.45% Tween 20) and covered with mineral oil. PCR reaction was carried out with 5min denature at 94℃, and then entered into 35 cycles. One cycle included: 30sec denature at 95℃, 1min annealing at 55℃, 1min and 30sec extension at 72℃, and finally 10min extension at 72℃.
Two μl PCR product was added into 2μl dilution containing 95% formamide, 20mmol/L EDTA, and 0.05% bromophenol blue/0.05% xylene cyanol dye. The mixture was denatured 5min at 95℃, then applied to a 6% polyacrylamide gel (21cm×40cm×0.4cm) with 1×TBE buffer at 45mA current and 45 Watt in cold room (4℃) for 4-5 hours. The gel was dried at 80℃ and autoradiographed at -70℃.

RESULTS
Pathohistology
All tumor specimens were reviewed. They were all hepatocellular carcinomas pathohistologically. The Fourteen (50%) cases of adjacent liver tissues had chronic hepatitis of active type (CAH) and persistent type (CPH), 6 cases had liver cirrhosis with CAH, 6 had liver cirrhosis and 2 cases had acute hepatitis.

Point mutation of N-ras gene
The result of PCR-SSCP for N-ras showed only 3 bands in normal control samples including human liver tissues (2 cases) and human placenta DNA (1 case). These 3 normal bands were also clearly discerned in all HCC and their adjacent tissues. Some of the detected specimens presented abnormal mobility shift bands. These bands demonstrated the point mutation in N-ras gene. The aberration rates at codon 2-37 of N-ras were 79.31% of HCCs and 80.77% of adjacent non-tumorous liver tissues. There was no significant difference between them (χ² test, P>0.05). Twenty-two of 29 HCC cases (75.86%) showed 2-5 abnormal mobility shifts bands.

HBV markers detection
HBsAg and HBxAg were localized in the cytoplasm. Some HBsAg were localized on cell membrane. HBsAg positive rate was 86.2% (25/29), and that of HBxAg was 79.3% (23/29). Statistically significant correlation (P<0.01) was observed in positive rate between HBsAg and HBxAg.

DISCUSSION
A lot of researches indicated that the patients with chronic hepatitis B had higher risk of developing HCC than uninfected population^[5,6] . HBxAg may be produced from a viral template integrated into the host genome during chronic infection and could transactivate oncogenes^[7] . In this study, the positive rates of HBsAg and HBxAg were 86.2% and 79.3%. There was a close relationship between HBxAg and HBV infection statistically (P<0.01). The results also suggested that HCC was closely associated with HBxAg.
PCR-SSCP analysis is based on that single-strand DNAs of the same nucleotide length in which the nucleotide sequences differed at only one or some positions can be separated by polyacrylamide gel electrophoresis. A variety of positions in a fragment of DNA polymorphisms could cause differences in the conformation of DNA chain and result in mobility shift of the single strands on gel electrophoresis.
In HCC, N-ras was firstly proved as one of the transforming genes^[4] , which belongs to G protein family. When it is converted to active oncogene by point mutation, chromosome rearrangement or gene amplification, the signal transmission of cell membranes may change, which drives cell division, resulting in abnormal differentiation and finally neoplasm. Enhanced expression of N-ras gene in human HCC has been reported^[3] . The “hot spot” mutation at codon 12, 13 or 61 in the N-ras gene have been described in many kinds of human cancers^[5] . But no point mutation around these “hot spots” was found in HCC from South Africa^[6] and Japan^[5] . In China, FANG Dian-Chun found that 37.2% (16/43) of HCC showed N-ras gene point mutation at codon 12 by PCR-restriction fragment length polymorphisms (PCR-RFLP). In our study, the PCR product contained codon 12, 13 of N-ras gene and the point mutation rate of this gene was 79.31% (23/29) of HCC, indicating a rather high frequency of N-ras gene mutation in HCC in Guangxi region. In contrast to the reports about no N-ras gene point mutation or only one mutation at codon 12 in HCC, we found that 75.86% (22/29) of HCC had 2-5 point mutations around codon 2-37. This showed that the mutation positions were not limited at codon 12 or 13. Therefore, if we only investigate the “hot spot” mutation at codon 12 of N-ras gene, there is a possibility of missing point mutations in other regions of this gene. The mobility shifts of eletrophoresis scattered at different positions. No clustering position could be considered as a hot spot mutation in this study. We do not know the exact codon of mutation in this fragment by PCR-SSCP analysis. They could be confirmed by DNA sequencing.
SSCP bands can be clearly discerned in HCC (79.31%) as well as their adjacent liver tissues (80.77%). There was no significant difference between them (P>0.05). Most of these non-tumorous liver tissues presented chronic hepatitis and/or liver cirrhosis, which was suggested to be closely correlated with HCC. It is indicated in our study that N-ras gene mutations were involved in the carcinogenesis and development of HCC.
Up to date, the mechanism of N-ras gene point mutation still remains unclear. FANG Dian-Chun et al found that mutation rate of ras gene in serum the HBsAg-positive group was 20% higher than that in HBsAg-negative group (P<0.05), which indicated that HBV infection may contribute to this mutation. A recent experiment gave the evidence that HBxAg may play a role in hepatocellular tumorigenesis by activating ras protein in vivo, forming ras-GTP complex and rapidly inducing increased cell replication^[12] . In this study, we observed that the detection of HBsAg (86.2%) and HBxAg (79.3%) was as high as that of N-ras mutation (79.3%). There was a parallel tendency between the detections of HBV markers and N-ras mutation. It has been reported that N-ras gene mutations occurred in rat HCC induced by aflatoxin B1 (AFB1)^[13] . The mutated points include codon 8, 13, 14 and 18. We found mutations of N-ras gene at multiple codons in Guangxi HCC. Previous epidemiological data showed that food AFB1 contamination is serious in Southern Guangxi. So, the N-ras gene mutation in Guangxi HCC may result from not only HBV infection, but also AFB1.

¹ Guangxi Cancer Institute, Nanning 530021, Guangxi Autonomous Region, China
² Institute of Liver Studies, King′s College Hospital, London, UK
Professor LIU Qi-Fu, male, born on 1929-11-28 in Guilin City, Guangxi Autonomous Region, China, graduated from Guangxi Medical College in 1954, working as a visiting scholar in University of Arkansas for Medical Sciences and University of Texas Medical School of USA between 1981-1983, now professor of biochemistry, Honorary Director of Guangxi Cancer Institute, tutor of postgraduates, majoring tumor biochemistry and molecular biology, having 30 papers published.
^* Project supported by the National Natural Science Foundation of China, No.39060032.
Correspondence to: Professor LIU Qi-Fu, Guangxi Cancer Institute, Nanning 530021, Guangxi Autonomous Region, China
Tel. +86*771*5313022 ext 3005， Fax. +86*771*5312523
Received 1998-01-31

REFERENCES
1 Miyaki M, Sato C, Gotanda T, Matsui T, Mishiro S, Imai M et al. Integration of region X of hepatitis B virus genome in human primary hepatocellular carcinomas propagated in nude mice. J Gen Virol, 1986;67(Pt7):1449-1454
2 Gu JR, Hu LF, Wan DF, Tian PK, Guo C, Huang LH. Study on N-ras gene—The human primary liver cancer transformation gene. Tumor, 1985;5(2):52-55
3 Li SY, Wang MS, Li SE. Expression of cellular oncogenes during hepatocarcinogenesis in rats. Acta Acad Med Sinica, 1990;12(2):121-124
4 Farshid M, Tabor E. Expression of oncogenes and tumor suppressor genes in human hepatocellular carcinoma and hepatoblastoma cell lines. J Med Virol, 1992;38(4):235-239
5 Breasley RP, Hwang LY, Lin CC, Chien CS. Hepatocellular carcinoma and hepatitis B virus: A prospective study of 22,707 men in Taiwan. Lancet, 1981;2(8256):1129-1133
6 Yeh FS, Yu MC, Mo CC, Luo S, Tong MJ, Henderson BE. Hepatitis B virus, aflatoxins, and hepatocellular carcinomas in Southern Guangxi, China. Cancer Res, 1989;49(9):2506-2509
7 Wang WL, London WT, Feitelson MA. Hepatitis B x antigen in hepatitis B virus carrier patients with liver cancer. Cancer Res, 1991;51(18):4971-4977
8 Tada M, Omata M, Ohto M. Analysis of ras gene mutations in human hepatic malignant tumors by polymerase chain reaction and direct sequencing. Cancer Res, 1990;50(4):1121-1124
9 Rechard CA, Short SA, Thorgeirsson SS, Huber BE. Characterization of a transforming N-ras gene in human hepatoma cell line HepG2: additional evidence for the importance of c-myc and ras cooperation in hepatocarcinogenesis. Cancer Res, 1990;50(5):1521-1527
10 Fang DC, Luo YH, Lu R, Xiao WH, Lin WW, Men RP et al. Studies of the point mutation of ras oncogene and suppressor gene p53 in 43 cases of hepatocellular carcinoma. Acta Acad Med Milit Tertlae, 1994;16(5):340-343
11 Leon M, Kew MC. Analysis of ras gene mutations in hepatocellular carcinoma in Southern African blacks. Anticancer Res, 1995;15(3):859-861
12 Doria M, Klein N, Lucito R, Schneider RJ. The hepatitis B virus HBx protein is a dual specificity cytoplasmic activator of Ras and nuclear activator of transcription factors. EMBO J, 1995;14(19):4747-4757
13 McMahon G, Davis EF, Huber LJ, Kim Y, Wogan GN. Characterization of c-ki-ras and N-ras in aflatoxin B1-induced rat liver tumors. Proc Natl Acad Sci USA, 1990;87(3):1104-1108