Identification of differentially expressed genes in hepatocellular carcinoma using representational difference analysis

Abstract

9700 Background: Hepatocellular carcinome (HCC) is a primary malignant neoplasm of the liver found throughout the world and is especially prevalent where the incidences of chronic hepatitis B (HBV) and C (HCV) are high. Molecular approaches have recently revealed involvement of the genes such as p53, p21, p27, p73, p16, pRb, pTEN, and MAGE have been demonstrated to be associated with HCC. However, the molecular mechanism of HCC carcinogenesis and development still remains to be elucidated. In this study, to identify the genes specifically expressed in HCC, subtractive library using representational difference analysis (RDA) coupled to microarray hybridization was applied to HCC and background liver tissues. Methods: Primary HCC and corresponding noncancerous liver tissues were obtained with informed consent from 5 patients who underwent hepatectomy. To identify differentially expressed genes in human HCC, subtractive library by RDA was constructed in HCC and the corresponding background liver tissue. After TA cloning and random selection of subtractive library clones, they were used for microarray hybridization. Results: After screening, more than 50 HCC specific gene fragments were identified, including alpha fetoprotein, glypican-3, preferentially expressed antigen in melanoma (PRAME), fucosidase alpha, carcinoembryonic antigen (CEA), and topoisomerase II. We further characterized one of differentially expressed gene, glypican 3. In situ hybridization showed that glypican 3 was moderate to intensely expressed in HCC but not in normal hepatocytes. Northern blot and RT-PCR of glypican 3 gene in 7 hepatocellular/hepatoblastoma cell lines showed, HepG2 cells express high levels and Hep3B and SNU368 cells express moderate to low levels of glypican 3. Conclusions: Glypican 3 is specifically expressed in HCC and is a cell surface protein, it can be a useful diagnostic marker and be a good target molecule for gene therapy or immunotherapy in HCC. No significant financial relationships to disclose.