Analysis of liver diesease related gene expression using subtraction cloning

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The genetic basis of hepatocellular carcinoma (HCC) has not yet been fully understood. Although various methods have been developed to detect differentially expressed genes in malignant diseases, efficient analysis from clinical specimens is generally difficult to perform due to the requirement of a large amount of samples. In the present study, we analyzed differentially expressed genes with a small amount of human HCC samples using suppression subtractive hybridization (SSH). Total RNA were obtained from the hepatitis C virus-associated HCC and adjacent to non-HCC liver tissues. cDNA was synthesized using modified RT-PCR, and then tester cDNA was ligated with two different kinds of adaptors and hybridized with an excess amount of driver cDNA. Tester specific cDNA was obtained by suppression PCR and the final PCR product was subcloned and sequenced. Seven known genes (focal adhesion kinase, deleted in colon cancer, guanine binding inhibitory protein a, glutamine synthetase, ornithine aminotransferase, M130, and pepsinogen C) and two previously unknown genes as being overexpressed in HCC, and 1 gene (decorin) as suppressed in HCC. Quantitative analysis of gene expression using quantitative RT-PCR demonstrated the differential expression of these genes in the original and other HCC samples. These findings demonstrated that it is possible to identify the previously unknown, differential gene expression from a small amount of clinical samples. Information about such alterations in gene expression could be useful for elucidating the genetic events in HCC pathogenesis, developing the new diagnosic markers, or determining novel therapeutic targets. Using similar technique, we found overexpression of IP10 in autoimmune hepatitis and chronic hepatitis C. In PBC, several mitochondrial genes are up-regulated.