| Summary: | 博士 === 國防醫學院 === 生命科學研究所 === 92 === Hepatocellular carcinomas (HCC) is the fifth most common cancers and the one of the ten major morality causes in Taiwan. The main purpose of this approach is to explore the molecular mechanisms of hepatocarcinogenesis. Using the methodology of functional genomics, I try to find out the genes induced the cellular transformation and to understand their tumorigenic mechanisms of HCC.The ability of anchorage independent growth (AIG) in soft agar is a hallmark of tumorigenicity in vitro. For isolation of novel cellular transforming genes that potentially participated in hepatocarcinogenesis, I conducted AIG assays on 10 human liver cancer cell lines and observed strong AIG capabilities in PLC5 and Huh7 but negligible in Tong cells. After cloning of genes by differential subtractive chain reactions (DSC) from strong AIG to AIG negative cells, I sequenced 2304 clones and identified 245 genes. The 245 genes were further evaluated by the following four stringent criteria including the gene location in amplified HCC genome reported by CGH, increased intensity in AIG positive cells by reverse Northern assays, multiple clones revealed by DSC subtractive cloning and sequencing, and overexpressed in more than 30% of 10 HCC tissues by small-scale quantitative RT-PCR. After four stringent criteria for selection of transforming genes among DSC clones, my results of quantitative RT-PCR analysis indicated that seven genes, DDX3, EIF3S2, CLIC1, HDGF, MST4, GPC3, and HSPCA were overexpressed in 64%, 62%, 60%, 58%, 53%, 49%, and 47%, respectively, of 45 HCC tissues. The results of cellular transformation capability by AIG assays indicated that the transfectants of EIF3S2 showed the strongest (>100-fold), DDX3, MST4 and CLIC1 were moderate, GPC3 and HSPCA were weak, and HDGF was none in forming colonies in soft agar. From the results in dissection of signaling pathways, the phosphorylation of STAT3 pathway is probably more important than that of PI3K/Akt in induction of transformation activity in Tong.
Together, our results suggested that Tong is a suitable human cell line for screening of overexpressed and/or cellular transforming genes. In addition, our results suggested that diverse functions of cellular transforming genes in various biological pathways could transform human Tong cells and potentially reveal new targets for drug development of HC
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