Gene expression profiles of hepatoma cell line HLE

Abstract

Aim: To investigate the global gene expression of cancer related genes in hepatoma cell line HLE using Atlas Human Cancer Array membranes with 588 well-characterized human genes related with cancer and tumor biology.

Methods: Hybridization of cDNA blotting membrane was performed with (32)P-labeled cDNA probes synthesized from RNA isolated from Human hepatoma cell line HLE and non-cirrhotic normal liver which was liver transplantation donor. AtlasImage, a software specific to array, was used to analyze the result. The expression pattern of some genes identified by Atlas arrays hybridization was confirmed by reverse transcription polymerase chain reaction (RT-PCR) in 24 pairs of specimens and Northern blot of 4 pairs of specimens.

Results: The differential expression of cell cycle/growth regulator in hepatocellular carcinoma (HCC) showed a stronger tendency toward cell proliferation with more than 1.5-fold up-regulation of Cyclin C, ERK5, ERK6, E2F-3, TFDP-2 and CK4. The anti-apoptotic factors such as Akt-1 were up-regulated, whereas the promotive genes of apoptosis such as ABL2 were down-regulated. Among oncogene/tumors suppressors, SKY was down-regulated. Some genes such as Integrin beta 8, Integrin beta 7, DNA-PK, CSPCP, byglycan, Tenacin and DNA Topo were up-regulated. A number of genes, including LAR, MEK1, eps15, TDGF1, ARHGDIA were down-regulated. In general, expression of the cancer progression genes was up-regulated, while expression of anti-cancer progression genes was down-regulated. These differentially expressed genes tested with RT-PCR were in consistent with cDNA array findings.

Conclusion: Investigation of these genes in HCC is helpful in disclosing molecular mechanism of pathogenesis and progression of HCC. For the first time few genes were discovered in HCC. Further study is required for the precise relationship between the altered genes and their correlation with the pathogenesis of HCC.

Figure 1

Parallel analyses of gene expression profiles in hu-man hepatoma cell line HLE and normal liver tissues. Atlas human cancer cDNA expression array (Clontech, USA) was hybridized with ³²P-labeled cDNA probes in normal liver tis-sues (A) and human hepatoma cell line HLE (B).

Figure 2

Partial semi-quantitative RT-PCR for 2 genes in 24 paired tissues. A total of 10 µl RT-PCR products were electro-phoresed on 2% agarose gel containing ethidium bromide. GAPDH was used as an internal control. (RT-PCR, reverse tran-scription polymerase chain reaction; N, adjacent normal liver tissue; C, human hepatocellular carcinoma tissue; GAPDH, glyceraldehyde-s-phosphate dehydrogenase; M, pUC Mix Maker).

Figure 3

Northern blot analysis of 2 genes to confirm the Atlas human cancer cDNA expression array. Four paired cases were used to determine these genes expression patterns. Twenty µg RNA was analyzed on a 1.2% denaturing agrose gel and trans-ferred onto a nylon membrane. ³²P-labeled cDNA probes for these genes were hybridized to the RNA-blotted membranes. After stringent washes, membranes were exposed to X-ray film overnight at -70 °C. The same membranes were rehybridized with human β-actin for an RNA loading control (N, adjacent normal liver tissue; C, human HCC tissue).