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Open Access Highly Accessed Research article

Quantitative evaluation of RASSF1A methylation in the non-lesional, regenerative and neoplastic liver

Sonia Di Gioia1, Paolo Bianchi1, Annarita Destro1, Fabio Grizzi2, Alberto Malesci36, Luigi Laghi13, Massimo Levrero5, Alberto Morabito6 and Massimo Roncalli46*

Author Affiliations

1 Laboratory of Molecular Genetics, Humanitas Clinical Institute of Rozzano, Milan, Italy

2 Scientific Direction, Humanitas Clinical Institute of Rozzano, Milan, Italy

3 Department of Gastroenterology, Humanitas Clinical Institute of Rozzano, Milan, Italy

4 Department of Pathology, Humanitas Clinical Institute of Rozzano, Milan, Italy

5 Laboratory of Gene Expression, Fondazione Andrea Cesalpino, University of Rome La Sapienza, Rome, Italy

6 University of Milan. Milan, Italy

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BMC Cancer 2006, 6:89  doi:10.1186/1471-2407-6-89

Published: 10 April 2006

Abstract

Background

Epigenetic changes during ageing and their relationship with cancer are under the focus of intense research. RASSF1A and NORE1A are novel genes acting in concert in the proapoptotic pathway of the RAS signalling. While NORE1A has not been previously investigated in the human liver, recent reports have suggested that RASSF1A is frequently epigenetically methylated not only in HCC but also in the cirrhotic liver.

Methods

To address whether epigenetic changes take place in connection to age and/or to the underlying disease, we investigated RASSF1A and NORE1A gene promoter methylation by conventional methylation specific PCR and Real-Time MSP in a series of hepatitic and non-hepatitic livers harboring regenerative/hyperplastic (cirrhosis/focal nodular hyperplasia), dysplastic (large regenerative, low and high grade dysplastic nodules) and neoplastic (hepatocellular adenoma and carcinoma) growths.

Results

In the hepatitic liver (chronic hepatitic/cirrhosis, hepatocellular nodules and HCC) we found widespread RASSF1A gene promoter methylation with a methylation index that increased from regenerative conditions (cirrhosis) to hepatocellular nodules (p < 0.01) to HCC (p < 0.001). In the non-hepatitic liver a consistent pattern of gene methylation was also found in both lesional (focal nodular hyperplasia and hepatocellular adenoma) and non-lesional tissue. Specifically, hepatocellular adenomas (HA) showed a methylation index significantly higher than that detected in focal nodular hyperplasia (FNH) (p < 0.01) and in non-lesional tissue (p < 0.001). In non-lesional liver also the methylation index gradually increased by ageing (p = 0.002), suggesting a progressive spreading of methylated cells over time. As opposed to RASSF1A gene promoter methylation, NORE1A gene was never found epigenetically alterated in both hepatitic and non-hepatitic liver.

Conclusion

We have shown that in non-lesional, regenerative and neoplastic liver the RASSF1A gene is increasingly methylated, that this condition takes place as an age-related phenomenon and that the early setting and spreading over time of an epigenetically methylated hepatocyte subpopulation, might be related to liver tumorigenesis.