Integrin gene expression profiles of human hepatocellular carcinoma

Abstract

Aim: To investigate gene expression profiles of integrin genes in hepatocellular carcinoma (HCC) through the usage of Atlas Human Cancer Array membranes, semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Northern blot.

Methods: Hybridization of cDNA array membrane was performed with alpha(32)P-labeled cDNA probes synthesized from RNA isolated from hepatocellular carcinoma and adjacent non-cirrhotic liver. AtlasImage, which is a software specific to array, was used to analyze the result. RT-PCR of 24 pairs specimen and Northern blot of 4 pairs specimen were used to confirm the expression pattern of some integrin genes identified by Atlas arrays hybridization.

Results: Among 588 genes spotted in membrane, 17 genes were related to integrin. Four genes were up-regulated, such as integrin alpha8, beta1, beta7 and beta8 in HCC. Whereas there were no genes down-regulated in HCC. RT-PCR and Northern blot analysis of integrin beta1 gene gave results consistent with cDNA array findings.

Conclusion: Investigation of these integrin genes should help to disclose the molecular mechanism of the cell adhesion, invasive and metastasis of HCC. A few genes are reported to have changed in HCC for the first time. The quick and high-throughout method of profiling gene expression by cDNA array provides us overview of key factors that may involved in HCC, and may find the clue of the study of HCC metastasis and molecular targets of anti-metastasis therapy. The precise relationship between the altered genes and HCC is a matter of further investigation.

Figure 1

Parallel analysis of intergrin gene expression in human hepatocellular carcinoma and adjacent normal liver tissues. Atlas human cancer cDNA expression array (Clontech, USA) is hybridized with ³²P-labeled cDNA probes obtained by RT-PCR from total RNA of human hepatocellular carcinoma (A) and adjacent normal liver tissues (B). The intergrin gene region is marked in the photo. The colorful comparative diagram between human hepatocellular carcinoma and adjacent normal liver tissues is obtained when one aligns two arrays to AtlasImage Grid Temples and adjusts the alignments and backgroud calculations (C). Definitions of colors in the array comparison are showed (D).

Figure 2

Partial semi-quantitative RT-PCR results for Intergrin beta1 in 24 paired tissues. A total of 10 μL RT-PCR products are electrophoresed on 2% agarose gel containing ethidium bromide. The level of GAPDH is used as an internal control. (RT-PCR, reverse transcription- polymerase chain reaction; N, adjacent noamal liver tissue; C, human hepatocellular carcinoma tissue; GAPDH, glyceraldehyde-s-phosphate dehydrogenase; M, pUC Mix Maker)

Figure 3

Nothern blot analysis of Intergrin beta1, which differentially express in human hepatocellular carcinoma and adjacent normal liver tissues, to confirm the Atlas human cancer cDNA expression array. Four paired cases are used to determine these genes expression patterns. Twenty μg RNA is analyzed on a 1.2% denaturing agrose gel and tansfer onto a nylon membrane. ³²P-labeled cDNA probes for these genes are hybridized to the RNA-bloted membranes. After stringent washes, membranes are exposed to X-ray film overnight at -70 °C. The same membranes are rehybridized with human β-actin for an RNA loading control. (C, human hepatocellular carcinoma tissue; N, adjacent noamal liver tissue)