Brief Report
Copyright ©The Author(s) 2001. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Aug 15, 2001; 7(4): 579-582
Published online Aug 15, 2001. doi: 10.3748/wjg.v7.i4.579
Cloning of differentially expressed genes in human hepatocellular carcinoma and nontumor liver
Xiao-Yan Cao, Jie Liu, Zhao-Rui Lian, Marcy Clayton, Jia-Lu Hu, Ming-Hua Zhu, Dai-Ming Fan, Mark Feitelson
Xiao-Yan Cao, Jie Liu, Jia-Lu Hu, Dai-Ming Fan, Institute of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an 710033, Shaanxi Province, China
Zhao-Rui Lian, Marcy Clayton, Mark Feitelson, Department of Pathology & Cell Biology, Thomas Jefferson University, Philadelphia, PA19107 USA
Ming-Hua Zhu, Department of Pathology, Second Military Medical University, Shanghai 200433, China
Author contributions: All authors contributed equally to the work.
Supported by NSFC Grant 30024002.
Correspondence to: Jie Liu, Institute of Digestive Diseases, Xijing Hospital, Xi’an 710033, Shaanxi Province, China.
Telephone: 0086-29-3375229 Fax: 0086-29-2359041
Received: April 6, 2001
Revised: May 11, 2001
Accepted: May 18, 2001
Published online: August 15, 2001

Key Words: liver neoplasms/virology, carcinoma, hepatocellular/virology, hepatitis B virus/genetics, genes, viral, gene expression, cloning, molecular


The mechanism of hepatocellular carcinoma (HCC) is still unclear, although some genes have been found to play a role in the transformation of liver cells, and a variety of studies have described differences in gene expression which distinguished tumor from nontumor[1-6]. The new genes, especially the functional genes directly related with tumor are still worth being found.

The purpose of our study is to find the different genes between human liver tumor and normal tissues using suppression subtractive hybridization.

Patients samples

HCC and surrounding nontumor liver tissues used for analysis were obtained from the patients who had undergone surgery for the removal of their tumors in Xijing Hospital. Fresh frozen blocks and -80 °C snap frozen paired liver and tumor samples from individual patients were collected, and were then made available for RNA extraction and in situ hybridization.

PCR selected cDNA subtraction, cloning, sequencing and identification of cloned gene fragments

The difference in gene expression between human tumor and nontumor tissues were evaluated by a commercially available subtraction hybridization approach (the PCR selected cDNA subtraction kit from Clontech, Palo Alto, CA, USA) according to the instruction provided by the manufacturer. Briefly, we got total RNA and mRNA from tumor and nontumor tissues using the Qiagen RNeasy Kit (Qiagen, Inc. Valencea, CA, USA), and then both mRNA (2 μg each) were converted into cDNA. We refer to the cDNA from tumor as tester, and the reference cDNA from nontumor as driver. The tester and driver cDNA were digested with Rsa I to obtain shorter, blunt-ended moleclule. The tester cDNA was then subdivided into two portions and each ligated with different cDNA adapters. The driver cDNA had no adaptor. Two hybridization was then performed. In the first hybridization, an excess of driver cDNA was added to each sample of tester for equalization and enrichment of differentially expressed gene. During the second hybridization, templates for PCR amplification were generated from differentially expressed sequence. The entire population of molecules was then subjected into PCR to amplify the desired differentially expressed genes. In the first PCR, only differentially expressed genes were amplified exponentially because of using suppression PCR. The second PCR was performed using nested primer to reduce any background and to further enrich differentially expressed genes. The cDNA fragments were directly inserted into a T/A cloning vector(Novagen, Medison, WI, USA), and homology analysis was undertaken within GeneBank. On the other hand, we used normal tissues as the tester and tumor as the driver to do PCR select cDNA hybridization. The procedure was as above.

In situ hybridization (ISH)

The gene fragments obtained from PCR select cDNA subtraction were used as probes for in situ hybridization (ISH). ISH was conducted to verify that the subtraction hybridization procedure yielded probes whose expression differed in tumor compared to normal tissue. ISH was carried out using the Oncor ISH and digoxigenenin/biotin detection kits according to the instruction provided by the manufacturer (Oncor, Gaithersburg, MD, USA).

PCR selected cDNA subtraction, cloning, sequencing and GeneBank search

PCR select cDNA subtraction generated totally 19 differentially expressed genes in tumors and nontumors. Among them, 14 cDNA fragments had considerable homology with known genes in GeneBank (Table 1). For example, T2 and T3 had homology with ribosomal protein and elongation factor EF-1α, suggesting that these genes may stimulate cell growth. N1 from normal tissues had homology with interferon gamma gene, suggesting that this gene may be a negative regulator for cell growth. Interestingly, one gene from tumor and three genes from normal liver tissues had no homology as compared with those in GeneBank, which implied that these may be new genes.

Table 1 Differentially expressed genes in human tumor and nontumor liver.
CloneGeneBank search
Match% homology
T1Retinoblastoma gene (L11910)75% in 193 bp overlap
T2Ribosomal protein L7 (L16588)87% in 209 bp overlap
T3Elongation factor EF-1α (J04617)85% in 157 bp overlap
T42-oxoglutarate dehydrogenase (D10525)89% in 258 bp overlap
T5Proteasome activator HPA28 subunit β (D45348)93% in 204 bp overlap
T6Ribosomal protein S2 (X57432)89% in 195 bp overlap
T7Rab geramylgeranyl transferase-α Subunit (Y08200)90% in 110 bp overlap
T8Nuclear-encoded mitochondrial NADH-ubiquitinone reductase93% in 197 bp overlap
N1Interferon gamma gene (L07633)88% in 308 bp overlap
N3V-fos transformation effector protein92% in 200 bp overlap
N4Sigma-1 receptor (266537)75% in 123 bp overlap
N5Glycoprotein gll gene (D00464)- 3’flanking region62% in 549 bp overlap
N7RABAPTIN-5 protein (X91141)86% in 110 bp overlap
N8Dishevelled-3 (DUL3) protein89% in 72 bp overlap
Validation and in vivo expression patterns of these genes

The cDNA fragments obtained from subtraction hybridization of tumor and nontumor tissues were then used as probes for in situ hybridization. In all cases, the probes from tumor showed transcripts that were preferentially expressed in tumor tissues as compared with nontumors. In contrast, the genes from nontumor tissues demonstrated strong hybridization in normal tissues, but little or no signal in tumor tissues.


Hepatocellular carcinoma is one of the major causes of death in the world[7-10]. The mechanism of carcinogenesis is unknown, although it is widely accepted that hepatitis B virus (HBV) and hepatitis C virus (HCV) are closely related to liver cancer, especially hepatitis B virus X antigen[11-14]. A common feature of HBV infection is the integration of HBV DNA, in whole or in part, into host chromatin[15-17]. The sites of HBV integration are scattered throughout the host genome[18], making it unlikely that HBV brings about hepatocellular transformation by cis-acting mechanisms in most cases. With regard to virus sequences, integration commonly occurs within a small region near the end of the virus genome[19], which is consistent with the hypothesis that transformation may be associated with the expression of one or more virus proteins from the integrated templates acting in trans. Integrated fragments of HBV DNA have been shown to make a truncated preS/S and or HBX polypeptides, both of which have trans-activting activities[20-24]. However, only HBxAg transforms a mouse hepatocyte cell line in culture[25,26], and gives rise to liver tumors in at least one strain of transgenic mice[27-29]. Independent work has also shown that HBxAg stimulates the cell cycle, perhaps by the activation of a number of signal transduction pathways[30-34]. The expression of HBxAg is more consistent than that of preS in the liver of infected patients. In addition, the findings that HBxAg binds to and inactivates the tumor suppressor p53 both in vitro and in vivo[35-37], and that it may bind to and alter the function of other transcriptional factors in the cells[38], implied that HBxAg function is important to the pathogenesis of HCC. There is some evidence that HBxAg naturally trans-activates the insulin-like growth factor-1 (IGF-1) receptor[39], and may also stimulate the production of IGF-11[40], both of which may help sustain the survival and/or growth of tumor cells.

Because the mechanism of HCC induced by HBV still need to be elucidated, cloning of the genes, especially the genes associated with HBV and HCV, is still very important to account for the development of liver cancer. By the newly created method, which is the suppression subtractive hybridization, we identified the difference in gene expression which distinguished tumor from nontumor. The use of these fragments as probes for in situ hybridization of tumor and nontumor tissues verified that the PCR-selected cDNA subtraction actually yielded differences in the gene expression that distinguished tumor from nontumor, and that its differential expression may be relevant to the pathogenesis of HCC. It is not known whether these differences are associated with HBxAg associated trans-activation[41,42], its inhibition of protesome function[43] its ribo/deoxy APTase[44], or AMP kinase activation[45], and/or its ability to alter signal transduction pathways[46], because hepatitis B virus is closely associated with the development of chronic liver diseases, such as hepatitis and cirrhosis, as well as with the development of hepatocellular carcinoma (HCC)[47-60]. However, experiments are in progress to firmly address these issues.

The results of this study showed that the up-regulation of multiple genes in tumor which have considerable homology with known products from GeneBank, for example, ribasomal protein and elongation factor EF-12, suggesting that the function of these genes is likely to positively regulate cell growth. Several genes are generated from normal tissues and one has > 88% homology with interferon gamma gene, suggesting that these genes may be the negative regulators for cell growth. In addition, one gene from tumor and three genes from normal liver tissues had no homology, as compared with entries in GeneBank, which implied that these may be new genes, and that it is very important to clone the full-length genes of these cDNA fragments to do the functional analysis. This kind of experiments are already on the way.


Prof. Bo Rong Pan in Oncology Center, Xijing Hospital has made great contribution to this article.


Edited by Wu XN and Ma JY

1.  Begum NA, Mori M, Matsumata T, Takenaka K, Sugimachi K, Barnard GF. Differential display and integrin alpha 6 messenger RNA overexpression in hepatocellular carcinoma. Hepatology. 1995;22:1447-1455.  [PubMed]  [DOI]
2.  Darabi A, Gross S, Watabe M, Malafa M, Watabe K. Differential gene expression in experimental hepatocellular carcinoma induced by woodchuck hepatitis B virus. Cancer Lett. 1995;95:153-159.  [PubMed]  [DOI]
3.  Inui Y, Higashiyama S, Kawata S, Tamura S, Miyagawa J, Taniguchi N, Matsuzawa Y. Expression of heparin-binding epidermal growth factor in human hepatocellular carcinoma. Gastroenterology. 1994;107:1799-1804.  [PubMed]  [DOI]
4.  Wu GS, Kar S, Carr BI. Identification of a human hepatocellular carcinoma-associated tumor suppressor gene by differential display polymerase chain reaction. Life Sci. 1995;57:1077-1085.  [PubMed]  [DOI]
5.  Yamashita N, Ishibashi H, Hayashida K, Kudo J, Takenaka K, Itoh K, Niho Y. High frequency of the MAGE-1 gene expression in hepatocellular carcinoma. Hepatology. 1996;24:1437-1440.  [PubMed]  [DOI]
6.  Ueki T, Fujimoto J, Suzuki T, Yamamoto H, Okamoto E. Expression of hepatocyte growth factor and its receptor c-met proto-oncogene in hepatocellular carcinoma. Hepatology. 1997;25:862-866.  [PubMed]  [DOI]
7.  Tang ZY. Advances in clinical research of hepatocellular carci-noma in China. World J Gastroenterol. 1998;4:4-7.  [PubMed]  [DOI]
8.  Roberts LR, LaRusso NF. Potential roles of tumor suppressor genes and microsatellite instability in hepatocellular carcinogenesis in southern African blacks. World J Gastroenterol. 2000;6:37-41.  [PubMed]  [DOI]
9.  Schmid R. Prospect of gastroenterology and hepatology in the next century. World J Gastroenterol. 1999;5:185-190.  [PubMed]  [DOI]
10.  Yip D, Findlay M, Boyer M, Tattersall MH. Hepatocellular carcinoma in central Sydney: a 10-year review of patients seen in a medical oncology department. World J Gastroenterol. 1999;5:483-487.  [PubMed]  [DOI]
11.  Bian JC, Shen FM, Shen L, Wang TR, Wang XH, Chen GC, Wang JB. Susceptibility to hepatocellular carcinoma associated with null genotypes of GSTM1 and GSTT1. World J Gastroenterol. 2000;6:228-230.  [PubMed]  [DOI]
12.  Martins C, Kedda MA, Kew MC. Characterization of six tumor suppressor genes and microsatellite instability in hepatocellular carcinoma in southern African blacks. World J Gastroenterol. 1999;5:470-476.  [PubMed]  [DOI]
13.  Wei HS, Li DG, Lu HM. Hepatic cell apoptosis and fas gene. Shijie Huaren Xiaohua Zazhi. 1999;7:531-532.  [PubMed]  [DOI]
14.  Ning XY, Yang DH. Research and progress in vivo gene therapy for primary liver cancer. Shijie Huaren Xiaohua Zazhi. 2000;8:89-90.  [PubMed]  [DOI]
15.  He P, Tang ZY, Ye SL, Liu BB. Relationship between expression of alpha-fetoprotein messenger RNA and some clinical parameters of human hepatocellular carcinoma. World J Gastroenterol. 1999;5:111-115.  [PubMed]  [DOI]
16.  Sun HC, Li XM, Xue Q, Chen J, Gao DM, Tang ZY. Study of angiogenesis induced by metastatic and non-metastatic liver cancer by corneal micropocket model in nude mice. World J Gastroenterol. 1999;5:116-118.  [PubMed]  [DOI]
17.  Luo YQ, Wu MC, Cong WM. Gene expression of hepatocyte growth factor and its receptor in HCC and nontumorous liver tissues. World J Gastroenterol. 1999;5:119-121.  [PubMed]  [DOI]
18.  Yang JM, Han DW, Liang QC, Zhao JL, Hao SY, Ma XH, Zhao YC. Effects of endotoxin on expression of ras, p53 and bcl-2 oncoprotein in hepatocarcinogenesis induced by thioacetamide in rats. China Natl J New Gastroenterol. 1997;3:213-217.  [PubMed]  [DOI]
19.  Zhao GQ, Xue L, Xu HY, Tang XM, Hu RD, Dong J. In situ hybridization assay of androgen receptor gene in hepatocarcinogenesis. World J Gastroenterol. 1998;4:503-505.  [PubMed]  [DOI]
20.  Ma ZY, Fan QS, Zhang DF. The effect of acupuncture on the IL2-IFN-NKC immunoregulatory system of mice with HAC grafting hepatocarcinoma. World J Gastroenterol. 2000;6:32.  [PubMed]  [DOI]
21.  Li WJ, Gao QX, Zhou GM, Wei ZQ. Micronuclei and cell survival in human liver cancer cells irradiated by 25MeV/u (40)Ar14(+). World J Gastroenterol. 1999;5:365-368.  [PubMed]  [DOI]
22.  Liu LX, Jiang HC, Zhu AL, Zhou J, Wang XQ, Wu M. Gene expression profiles in liver cancer and normal liver tissues. World J Gastroenterol. 2000;6:85.  [PubMed]  [DOI]
23.  Jiang RL, Lu QS, Luo KX. Cloning and expression of core gene cDNA of Chinese hepatitis C virus in cosmid pTM3. World J Gastroenterol. 2000;6:220-222.  [PubMed]  [DOI]
24.  Zhang SZ, Liang JJ, Qi ZT, Hu YP. Cloning of the non-structural gene 3 of hepatitis C virus and its inducible expression in cultured cells. World J Gastroenterol. 1999;5:125-127.  [PubMed]  [DOI]
25.  Zhou XP, Wang HY, Yang GS, Chen ZJ, Li BA, Wu MC. Cloning and expression of MXR7 gene in human HCC tissue. World J Gastroenterol. 2000;6:57-60.  [PubMed]  [DOI]
26.  Assy N, Gong Y, Zhang M, Minuk G. Appearance of an inhibitory cell nuclear antigen in rat and human serum during variable degrees of hepatic regenerative activity. World J Gastroenterol. 1999;5:103-106.  [PubMed]  [DOI]
27.  Okuda K. Hepatocellular carcinoma: recent progress. Hepatology. 1992;15:948-963.  [PubMed]  [DOI]
28.  Matsubara K, Tokino T. Integration of hepatitis B virus DNA and its implications for hepatocarcinogenesis. Mol Biol Med. 1990;7:243-260.  [PubMed]  [DOI]
29.  Feitelson MA, Duan LX. Hepatitis B virus X antigen in the pathogenesis of chronic infections and the development of hepatocellular carcinoma. Am J Pathol. 1997;150:1141-1157.  [PubMed]  [DOI]
30.  Tiollais P, Pourcel C, Dejean A. The hepatitis B virus. Nature. 1985;317:489-495.  [PubMed]  [DOI]
31.  Dejean A, Sonigo P, Wain-Hobson S, Tiollais P. Specific hepatitis B virus integration in hepatocellular carcinoma DNA through a viral 11-base-pair direct repeat. Proc Natl Acad Sci USA. 1984;81:5350-5354.  [PubMed]  [DOI]
32.  Caselmann WH, Meyer M, Kekulé AS, Lauer U, Hofschneider PH, Koshy R. A trans-activator function is generated by integration of hepatitis B virus preS/S sequences in human hepatocellular carcinoma DNA. Proc Natl Acad Sci USA. 1990;87:2970-2974.  [PubMed]  [DOI]
33.  Kekulé AS, Lauer U, Meyer M, Caselmann WH, Hofschneider PH, Koshy R. The preS2/S region of integrated hepatitis B virus DNA encodes a transcriptional transactivator. Nature. 1990;343:457-461.  [PubMed]  [DOI]
34.  Zahm P, Hofschneider PH, Koshy R. The HBV X-ORF encodes a transactivator: a potential factor in viral hepatocarcinogenesis. Oncogene. 1988;3:169-177.  [PubMed]  [DOI]
35.  Wollersheim M, Debelka U, Hofschneider PH. A transactivating function encoded in the hepatitis B virus X gene is conserved in the integrated state. Oncogene. 1988;3:545-552.  [PubMed]  [DOI]
36.  Schlüter V, Meyer M, Hofschneider PH, Koshy R, Caselmann WH. Integrated hepatitis B virus X and 3' truncated preS/S sequences derived from human hepatomas encode functionally active transactivators. Oncogene. 1994;9:3335-3344.  [PubMed]  [DOI]
37.  Höhne M, Schaefer S, Seifer M, Feitelson MA, Paul D, Gerlich WH. Malignant transformation of immortalized transgenic hepatocytes after transfection with hepatitis B virus DNA. EMBO J. 1990;9:1137-1145.  [PubMed]  [DOI]
38.  Seifer M, Höhne M, Schaefer S, Gerlich WH. In vitro tumorigenicity of hepatitis B virus DNA and HBx protein. J Hepatol. 1991;13 Suppl 4:S61-S65.  [PubMed]  [DOI]
39.  Kim CM, Koike K, Saito I, Miyamura T, Jay G. HBx gene of hepatitis B virus induces liver cancer in transgenic mice. Nature. 1991;351:317-320.  [PubMed]  [DOI]
40.  Koike K, Moriya K, Iino S, Yotsuyanagi H, Endo Y, Miyamura T, Kurokawa K. High-level expression of hepatitis B virus HBx gene and hepatocarcinogenesis in transgenic mice. Hepatology. 1994;19:810-819.  [PubMed]  [DOI]
41.  Ueda H, Ullrich SJ, Gangemi JD, Kappel CA, Ngo L, Feitelson MA, Jay G. Functional inactivation but not structural mutation of p53 causes liver cancer. Nat Genet. 1995;9:41-47.  [PubMed]  [DOI]
42.  Benn J, Schneider RJ. Hepatitis B virus HBx protein activates Ras-GTP complex formation and establishes a Ras, Raf, MAP kinase signaling cascade. Proc Natl Acad Sci USA. 1994;91:10350-10354.  [PubMed]  [DOI]
43.  Natoli G, Avantaggiati ML, Chirillo P, Costanzo A, Artini M, Balsano C, Levrero M. Induction of the DNA-binding activity of c-jun/c-fos heterodimers by the hepatitis B virus transactivator pX. Mol Cell Biol. 1994;14:989-998.  [PubMed]  [DOI]
44.  Benn J, Su F, Doria M, Schneider RJ. Hepatitis B virus HBx protein induces transcription factor AP-1 by activation of extracellular signal-regulated and c-Jun N-terminal mitogen-activated protein kinases. J Virol. 1996;70:4978-4985.  [PubMed]  [DOI]
45.  Cross JC, Wen P, Rutter WJ. Transactivation by hepatitis B virus X protein is promiscuous and dependent on mitogen-activated cellular serine/threonine kinases. Proc Natl Acad Sci USA. 1993;90:8078-8082.  [PubMed]  [DOI]
46.  Benn J, Schneider RJ. Hepatitis B virus HBx protein deregulates cell cycle checkpoint controls. Proc Natl Acad Sci USA. 1995;92:11215-11219.  [PubMed]  [DOI]
47.  Feitelson MA, Zhu M, Duan LX, London WT. Hepatitis B x antigen and p53 are associated in vitro and in liver tissues from patients with primary hepatocellular carcinoma. Oncogene. 1993;8:1109-1117.  [PubMed]  [DOI]
48.  Wang XW, Forrester K, Yeh H, Feitelson MA, Gu JR, Harris CC. Hepatitis B virus X protein inhibits p53 sequence-specific DNA binding, transcriptional activity, and association with transcription factor ERCC3. Proc Natl Acad Sci USA. 1994;91:2230-2234.  [PubMed]  [DOI]
49.  Truant R, Antunovic J, Greenblatt J, Prives C, Cromlish JA. Direct interaction of the hepatitis B virus HBx protein with p53 leads to inhibition by HBx of p53 response element-directed transactivation. J Virol. 1995;69:1851-1859.  [PubMed]  [DOI]
50.  Henkler F F, Koshy R. Hepatitis B virus transcriptional activators: mechanisms and possible role in oncogenesis. J Viral Hepat. 1996;3:109-121.  [PubMed]  [DOI]
51.  Kim SO, Park JG, Lee YI. Increased expression of the insulin-like growth factor I (IGF-I) receptor gene in hepatocellular carcinoma cell lines: implications of IGF-I receptor gene activation by hepatitis B virus X gene product. Cancer Res. 1996;56:3831-3836.  [PubMed]  [DOI]
52.  Su Q, Liu YF, Zhang JF, Zhang SX, Li DF, Yang JJ. Expression of insulin-like growth factor II in hepatitis B, cirrhosis and hepatocellular carcinoma: its relationship with hepatitis B virus antigen expression. Hepatology. 1994;20:788-799.  [PubMed]  [DOI]
53.  Cheong JH, Yi M, Lin Y, Murakami S. Human RPB5, a subunit shared by eukaryotic nuclear RNA polymerases, binds human hepatitis B virus X protein and may play a role in X transactivation. EMBO J. 1995;14:143-150.  [PubMed]  [DOI]
54.  Antunović J, Lemieux N, Cromlish JA. The 17 kDa HBx protein encoded by hepatitis B virus interacts with the activation domains of Oct-1, and functions as a coactivator in the activation and repression of a human U6 promoter. Cell Mol Biol Res. 1993;39:463-482.  [PubMed]  [DOI]
55.  Huang J, Kwong J, Sun EC, Liang TJ. Proteasome complex as a potential cellular target of hepatitis B virus X protein. J Virol. 1996;70:5582-5591.  [PubMed]  [DOI]
56.  de-Medina T, Haviv I, Noiman S, Shaul Y. The X protein of hepatitis B virus has a ribo/deoxy ATPase activity. Virology. 1994;202:401-407.  [PubMed]  [DOI]
57.  Benn J, Su F, Doria M, Schneider RJ. Hepatitis B virus HBx protein induces transcription factor AP-1 by activation of extracellular signal-regulated and c-Jun N-terminal mitogen-activated protein kinases. J Virol. 1996;70:4978-4985.  [PubMed]  [DOI]
58.  Kekulé AS, Lauer U, Weiss L, Luber B, Hofschneider PH. Hepatitis B virus transactivator HBx uses a tumour promoter signalling pathway. Nature. 1993;361:742-745.  [PubMed]  [DOI]
59.  Yu LC, Gu CH. Mutation of hepatitis B virus and its association with liver diseases. Shijie Huaren Xiaohua Zazhi. 1999;7:978-979.  [PubMed]  [DOI]
60.  Lau GKK. Immunological approaches to the breakdown of hepatitis B viral persistence. World J Gastroenterol. 1998;4:32.  [PubMed]  [DOI]