Study On The Differential Gene Expression Profiles During Hepatocarcinogenesis

Background and ObjectivesHepatocellular carcinoma (HCC) is one of the most common malignant primary liver tumors worldwide, especially in several areas of Asia and Africa. HCC is the fifth position at the incidence of malignant tumor and the third position at the cause of death in global. Recently, the incidence and mortality of HCC are increasing year by year. Hepatitis B virus (HBV) infection is suggested to be the main cause for chronic hepatitis, liver cirrhosis (LC) and HCC in China. HCC is the second cause of cancer death in our country, and the number of death is 110,000 per year. The development of HCC is hidden. In clinical, only less than 30% patients can be acquired the chance of operation. Although advances have been achieved in the diagnosis and treatment of HCC, there are so limited choice and poor prognosis for many middle and advanced stage of HCC patients. HCC is an aggressive malignancy which severely threatens the health and life of people. The development of HCC is a complex progress which multiple factors, multi-steps and multiple genes take part in. Generally speaking, hepatocarcinogenesis usually develops from chronic hepatitis and LC; However, the molecular mechanisms of hepatocarcinogenesis from chronic hepatitis and liver cirrhosis to HCC have not yet been clearly defined. Although it has been reported that a variety of abnormal expression of several genes might be involved in the developing pathological courses, such as mutation and deletion of tumor suppression genes, abnormal amplification and expression of oncogenes, but which genes are expressed differentially and specifically ,and how these roles in HCC are unknown. Traditional molecular study for single gene is limited and difficult to discover the hepatocarcinogenesis. New technology (gene chip) was developed recently for detecting gene expression profiles, which has advantages at high-throughput, microamount parallelisation and automation that can outshine traditional molecular technologies. Gene chip is a microarray device which tens of thousands oligonucleotide or cDNA fragments are fixed on a rigid surface (usually glass, nylon membrane, siice of silicon) with high-density. The oligonucleotide chip is the former. The principle of gene chip is the hybridization of nucleic acid. So gene chip technology can study the expression of tens of thousands genes accurately and efficiently at the same time in whole genome. It has already been an important method for tumors study. Here, we applied Affymetrix human genome plus 2.0 oligonucleotide chip containing 19378 known genes to research the gene expression profile in normal liver (NL), chronic hepatitis B (CHB), LC and HCC, and then, to analyze the common differentially expression of genes in four tissues and discuss the potential roles of these genes in the development of HCC.Materials and MethodsNine NL tissues, 6 LC and 15 HCC tissues infected by HBV were obtained from surgical specimens in Henan Provincial Hospital from March 2005 to June 2005. Thirteen CHB tissues were obtained from liver biopsy specimens in An Yang Fifth Hospital between February 2006 and April 2006. HBsAg is positive in CHB, LC and HCC, but negative in NL. All of the diseases are diagnosed by pathologic histology. After taking from the liver, the tissues were washed with nuclease-free PBS and frozen immediately in liquid nitrogen for using. The total RNA was isolated from frozen tissues according to the manual of Trizol kit (Invitrogen) and then purified based on the guide of RNeasy mini kit (Qiagen). The intensity ratio of 28S to 18S rRNA in total RNA samples was examined by 1% agarose gel electrophoresis (100V, 0.5 h) to estimate the total RNA integrity. According to the methods established by Affymetrix, cDNA and cRNA were synthesized. After that, cRNA was labeled with biotin, and then digested into 35-200 bp fragments. Affymetrix Human genome U133 plus2.0 chip was hybridized with biotin-labeled cRNA, and then stained with Affymetrix Genechip fluidics station 450. Finally, the chips were scanned by GeneChip scan 3000 7G system, and the signal values of gene expression were observed. The signals of gene expression on chips were obtained with GenePix Pro 3.0 software and quantified by normalizing the signal values. The signal ratio of gene expression was calculated based on standards (the signal of gene expression in CHB, LC, HCC are divided by corresponding signal of gene in NL). The criteria of screening differential genes in hepatocarcinogenesis: ratio value≥2 for up-regulated genes and <0.5 for down-regulated genes. Statistical analysis of data was done using Microsoft Excel and Gominer softwares. The information of HCC-related genes identified was further acquired from the NCBI (http://www.pubmed.com), GENEONTOLOGY databases (http://www.geneontology.org) and KEGG databases (http://www.biorcart.com).Results1. The integrity of RNA isolated in each group was fine. The ratio of absorbance of total RNA at OD260/280nm was between 1.8～2.0. Thermostability experiment at 70℃1h, 20℃1h showed there was no obvious RNA degradation. mRNA localized mainly at 0.9～4.0kb consecutive strip by electrophoresis, indicating that high quality of RNA was extracted.2. The results were completely in concord with the standards of Affymetrix U133 Plus 2.0 gene chip quality control. The signal intensity of probes met the standards of quality control. Normal detecting system ensured the reliability of our results.3. Compared with NL tissues, differential expression profiles of genes in CHB, LC and HCC were screened. Bioinformatics was used to analyze the common differential expression profiles of genes in three tissues. Eighty-one genes were screened out, which are involved with protooncogenes, tumor suppressor genes, cell signal transduction related genes, cell cycle related genes, metabolic enzymes genes, immune-related genes, tumor associated antigen genes and so on. Of the 81 genes, 53 genes were consistently up-regulated and 28 genes were consistently down-regulated. Sixty-six genes were known and 15 were unclear in terms of function. Conclusions1. Multiple genes take part in the carcinogenesis of HCC. The oligonucleotide chip for analysis of gene expression profile is a powerful method to screen HCC-related genes, and it can effectively analyzes the potential roles of these genes in the development of HCC.2. The abnormal expression of 81 genes which may be associated with liver carcinogenesis. These abnormal genes also expressed in CHB and LC , which may take part in the whole course of hepatocarcinogenesis.3. To analyses the 81 abnormal expressed genes can help to understand the mechanism of hepatocarcinogenesis at molecular level and offer clues for exploring new potential tumor markers for diagnosis and targets for gene therapy of HCC.4. Some of abnormal expression genes have not reported in the liver diseases. We need to further investigate the roles of these genes in the hepatocarcinogenesis.