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Promoter hypermethylation of Wnt pathway inhibitors in hepatitis C virus - induced multistep hepatocarcinogenesis

Muhammad Umer1, Sohail Asif Qureshi2, Zahid Yasin Hashmi34, Asif Raza4, Janbaz Ahmad5, Moazur Rahman1 and Mazhar Iqbal1*

Author Affiliations

1 Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad 38000, Pakistan

2 Syed Babar Ali School of Science & Engineering, Lahore University of Management Sciences, Sector-U, DHA, Lahore, Pakistan

3 Department of Medicine, Punjab Medical College, Faisalabad 38800, Pakistan

4 Liver Center, District Headquarter Hospital, Faisalabad 38000, Pakistan

5 Department of Pathology, Punjab Medical College, Faisalabad 38800, Pakistan

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Virology Journal 2014, 11:117  doi:10.1186/1743-422X-11-117

Published: 20 June 2014

Abstract

Background

Aberrant DNA methylation profiles are a characteristic feature of almost all types of cancers including hepatocellular carcinoma (HCC) and play an important role in carcinogenesis. In spite of the accumulating evidence that suggests appearance of such aberrations at precancerous stages, very little effort has been invested to investigate such possible methylation events in patients at risk of developing HCC i.e. those suffering from chronic hepatitis C virus (HCV) infection and liver cirrhosis (LC). We reasoned that such an analysis could lead to the identification of novel predictive biomarkers as well as potential drug targets.

Methods

Promoter methylation status of two Wnt inhibitors SFRP2 and DKK1 was quantitatively analyzed by bisulfite pyrosequencing in a series of liver biopsy samples. These biopsies were collected from HCV-infected individuals suffering from chronic hepatitis (CH; n = 15), liver cirrhosis (LC; n = 13) and hepatocellular carcinoma (HCC; n = 41). DNA isolated from infection free normal livers (N; n =10) was used as control.

Results

Our analysis revealed that both of the genomic loci were significantly hypermethylated in CH patients’ livers as compared to normal controls (p = 0.0136 & 0.0084 for SFRP2 and DKK1, respectively; Mann–Whitney U test). DNA methylation levels for both loci were also significantly higher in all the diseased cohorts as compared to normal controls (p < 0.0001 and = 0.0011 for SFRP2 and DKK1, respectively; Kruskal-Wallis test). However, a comparison between three disease cohorts (CH, LC & HCC) revealed no significant difference in levels of DNA methylation at DKK1 promoter. In contrast, a progressive increase in DNA methylation levels was observed at the SFRP2 promoter (i.e. N < CH & LC < HCC).

Conclusion

This study demonstrated that in HCV infected liver tissues hypermethylation at promoter regions of key cancer related genes SFRP2 and DKK1, appears early at CH and LC stages, long before the appearance of HCC.

Keywords:
Hypermethylation; Wnt pathway; HCV; SFRP2; DKK1; Hepatocarcinogenesis