RASSF1A And DOK1 Promoters Methylation Levels In Hepatic Tissues With And Without Hepatocellular Carcinoma

Oscar ARA┌JO, Oswaldo Cruz Foundation, Brazil
ROSA A. 1 , NIEL C. 1 , FERNANDES A. 2 , VILLELA-NOGUEIRA C. 3 , PANNAIN V. 2 , ARAUJO N. 1

1 Oswaldo Cruz Institute, Laboratory of Molecular Virology, Rio de Janeiro, Brazil.
2 Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro, Department of Pathology, Rio de Janeiro, Brazil.
3 Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro, Hepatology Division, Rio de Janeiro, Brazil.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. Liver cirrhosis promoted by chronic hepatitis C virus (HCV) and chronic hepatitis B virus (HBV) infections are the major risk factors for developing HCC. Hypermethylation of gene promoter regions is the main epigenetic silencing mechanism and has been associated with the development of HCC. Ras association domain family 1 isoform A (RASSF1A) and downstream of tyrosine kinase 1 (DOK1) are tumor suppressor genes whose inactivation has been reported in patients with HCC development. The aim of this study is to determine whether occurrence of aberrant methylation of RASSF1A and DOK1 is associated with the progression of liver disease and HCC in Brazilian patients. Methylation levels were measured by bisulfite pyrosequencing of DNA extracted from formalin-fixed, paraffin-embedded liver tissues. A total of 41 liver samples were analyzed. Of these, 20 were HCC, 9 cirrhotic and 12 normal tissues. In each sample, the percentage of methylation was determined for six RASSF1A and five DOK1 promoter CpG islands. A statistically significant difference (p< 0.001) was observed between HCC and non-HCC tissues for both genes. Mean percents of methylation in RASSF1A and DOK1 were as follows: 59.1% and 56% for HCC, 26.1% and 19.6% for cirrhotic, and 16.2% and 12% for normal tissues, respectively. In addition, hypermethylation for RASSF1A and DOK1 was found in 29% and 43% of the cirrhotic tissues, respectively, and in 88% of the HCC tissues for both genes. Our results suggest that methylation levels are related to the stage of the liver disease, being lower in normal, intermediate in cirrhotic, and higher in HCC tissues. This study will help to elucidate epigenetic changes associated to HCC that can be used as molecular markers for liver cancer prognosis, diagnosis and treatment.
Financial support: CNPq, FAPERJ and FIOCRUZ.