Indentification of Differrentially Expressed Genes in Human Normal Liver Cell Line between Under Normoxic and Hypoxic Condition by DD - PCR

Hypoxia occurs in various pathological conditions and this state in cells or tissues often leads to neovascularization. Especially, solid tumor growth, e.g., hepatocellular carinoma, is much related to angiogenesis and hypoxia is thought to be a main signal in solid tumor angiogenesis. To know the initial state of liver tumor, we investigated the contribution of hypoxic condition in normal liver using human normal liver cell line, Chang liver. To identify differentially expressed genes under normoxic (21% O₂ tension) or hypoxic (1% O₂ tension) condition, we used differential display of polymerase chain reaction (DD-PCR) technique using total RNA extracted from both of the condition. Five different polymerase chain reaction (PCR) primers were used to compare the gene expression patterns of cells under each condition. Our results revealed several up-regulated and down-regulated partial cDNA fragment. We isolated 10 different kinds of cDNA fragments which showed marked differences in two conditions for additional analysis. The DNA sequence homology matching analysed using CenBank and SWISS-Prot data base revealed six clones showed high similarities to the already known sequences. Among 6 clones, 2 clones shows sequence similarities with P. falciparum topoisomerase 1 gene whereas other clones to human 18S ribosomal protein (HKE3) mRNA, human mRNA for protein involved in DNA double-strand break repair and H^+/peptide cotransporter mRNA, respectively. Northern blot analysis using each clone as a probe, or RT-PCR confirmed that the differentially detected PCR products reflected the differential expression of each mRNA in each condition. These differentially detected genes might be related to liver tumorigenesis at early stage and will be a useful genes for analysing contribution of hypoxia to liver cancer.