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


CONCLUSIONS
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.
Di Gioia
Sonia
S
Department of Pathology, Humanitas Clinical Institute of Rozzano, Milan, Italy. sonia.digioia@unimi.it
Bianchi
Paolo
P
Destro
Annarita
A
Grizzi
Fabio
F
Malesci
Alberto
A
Laghi
Luigi
L
Levrero
Massimo
M
Morabito
Alberto
A
Roncalli
Massimo
M
D048868:Adaptor Proteins, Signal Transducing; D000236:Adenoma / Q000235:genetics / Q000473:pathology; D000328:Adult; D000368:Aged; D000375:Aging / Q000235:genetics / Q000378:metabolism; D051017:Apoptosis Regulatory Proteins; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor / Q000737:chemistry; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics; D044127:Epigenesis, Genetic; D005260:Female; D020518:Focal Nodular Hyperplasia / Q000235:genetics / Q000473:pathology; D016147:Genes, Tumor Suppressor; D006525:Hepatitis, Viral, Human / Q000235:genetics / Q000473:pathology; D022781:Hepatocytes / Q000737:chemistry / Q000473:pathology; D006801:Humans; D008099:Liver / Q000737:chemistry*; D008103:Liver Cirrhosis / Q000235:genetics / Q000473:pathology; D008107:Liver Diseases / Q000235:genetics* / Q000473:pathology; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology; D008115:Liver Regeneration / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D011230:Precancerous Conditions / Q000235:genetics / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics*
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D004273:DNA, Neoplasm; C411762:RASSF1 protein, human; C454042:RASSF5 protein, human; D025521:Tumor Suppressor Proteins; D020559:Monomeric GTP-Binding Proteins