Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis

Abstract

Background: Hepatocellular carcinoma (HCC) presents one of the major health threats in China today. A better understanding of the molecular genetics underlying malignant transformation of hepatocytes is critical to success in the battle against this disease. The methylation state of C5 of the cytosine in the CpG di-nucleotide that is enriched within or near the promoter region of over 50 % of the polymerase II genes has a drastic effect on transcription of these genes. Changes in the methylation profile of the promoters represent an alternative to genetic lesions as causative factors for the tumor-specific aberrant expression of the genes.

Methods: We have used the methylation specific PCR method in conjunction with DNA sequencing to assess the methylation state of the promoter CpG islands of twenty genes. Aberrant expression of these genes have been attributed to the abnormal methylation profile of the corresponding promoter CpG islands in human tumors.

Results: While the following sixteen genes remained the unmethylated in all tumor and normal tissues: CDH1, APAF1, hMLH1, BRCA1, hTERC, VHL, RARbeta, TIMP3, DAPK1, SURVIVIN, p14ARF, RB1, p15INK4b, APC, RASSF1c and PTEN, varying degrees of tumor specific hypermethylation were associated with the p16INK4a, RASSF1a, CASP8 and CDH13 genes. For instance, the p16INK4a was highly methylated in HCC (17/29, 58.6%) and less significantly methylated in non-cancerous tissue (4/29. 13.79%). The RASSF1a was fully methylated in all tumor tissues (29/29, 100%), and less frequently methylated in corresponding non-cancerous tissue (24/29, 82.75%).

Conclusions: Furthermore, co-existence of methylated with unmethylated DNA in some cases suggested that both genetic and epigenetic (CpG methylation) mechanisms may act in concert to inactivate the p16INK4a and RASSF1a in HCC. Finally, we found a significant association of cirrhosis with hypermethylation of the p16INK4a and hypomethylation of the CDH13 genes. For the first time, the survey was carried out on such an extent that it would not only provide new insights into the molecular mechanisms underscoring the aberrant expression of the genes in this study in HCC, but also offer essential information required for a good methylation-based diagnosis of HCC.

Figure 1

The methylation profiles of ten genes in human HCC patients (part 1). Both electrophoretic patterns of the representative PCR products of ten targets (indicated respectively at the top of figures) and the corresponding sequencing result of the one representative PCR product were presented. To indicate the methylation status, the sequenced data are aligned with the wild-type sequence. Representative samples are presented. * size marker. U, the unmethylated; and M, the methylated.

Figure 2

The methylation profiles of ten genes in human HCC patients (part 2). Both electrophoretic patterns of the representative PCR products of ten targets (indicated respectively at the top of figures) and the corresponding sequencing result of the one representative PCR product were presented. To indicate the methylation status, the sequenced data are aligned with the wild-type sequence. Representative samples are presented. * size marker. U, the unmethylated; and M, the methylated.

Figure 3

The methylation profiles of the p14^ARF and p15^INK4b genes in the hepatoma cell lines analyzed by both MSP and Southern analyses. Panel 1, The schematic illustration of the gene structure and the CpG islands of interests along with restriction digestion sites and the probes. The scale is arbitrary. Panel 2, The methylation profiling of the p14^ARF and p15^INK4b promoter CpG island by MSP. Panel 3, The schematic illustration of the CpG islands of p14^ARF with restriction digestion sites and the probes. Panel 4, The electrophoretic pattern of the digests before blotting of the genomic DNA. The cell origins of the genomic DNA are indicated at the top. The size markers are provided by Hind III digests of the lambda phage DNA and indicated in kb at the left side of the autorad. Abbreviations: B, BstX I; H: Hpa II, M: Msp I. Panel 5, The autorad of the Southern analysis hybridized by the probe indicated in panel 3. The size of the hybridized band is indicated at the left side of the antorad. Panel 6, The schematic illustration of the CpG islands of p14^ARF with restriction digestion sites and the probes. Panel 7, The electrophoretic pattern of the digests before blotting of the genomic DNA. The cell origins of the genomic DNA are indicated at the top. The size markers are provided by Hind III digests of the lambda phage DNA and indicated in kb at the left side of the autorad. Abbreviations: Hi: Hind III; H: Hpa II, M: Msp I. Panel 8, The autorad of the Southern analysis hybridized by the probe indicated in panel 6.