Abstract
BackgroundEpigenetic 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.MethodsTo 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.ResultsIn 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.ConclusionWe 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.
Highlights
Epigenetic changes during ageing and their relationship with cancer are under the focus of intense research
RASSF1A promoter methylation in hepatitic liver Qualitative analysis for the presence of methylated alleles by methylation-specific PCR (MSP) RASSF1A promoter methylation was detected by conventional MSP, in 100% (26/26) hepatocellular carcinomas (HCC), 86.6% (39/45) hepatocellular nodules (HN) and 81.1% (30/37) cirrhotic livers
DUFlipegpupearrnepe2al nsehlo: wRAinSgSFth1eA mpreotmhyolatetironrepgrioonfilwe iothf RCApSGSFi1sAlanddestescutbejdecbtyedMtSoPainnalHysCisC; uannddercloinreredsipsotnhdeinsegqcuirernhcoesriseported in the midUpper panel: RASSF1A promoter region with CpG islands subjected to analysis; underlined is the sequence reported in the middle panel showing the methylation profile of RASSF1A detected by MSP in HCC and corresponding cirrhosis
Summary
Epigenetic changes during ageing and their relationship with cancer are under the focus of intense research. Non neoplastic hepatitic/cirrhotic tissue usually adjacent to HCC, beside other methylated genes [18,19], frequently shows RASSF1A gene methylation ranging from 70% [13,15] to 82.75% [14], while non-neoplastic liver, or far from tumors, is not methylated [13,15,17]. These results suggest that RASSF1A methylation might be a potential marker of incipient malignancy in the human hepatocarcinogenesis
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