Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis.
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.
Yu
Jian
J
The State-Key Laboratory for Oncogenes and Related Genes, Shanghai Cancer Institute, LN 2200/25, Xie-Tu Road, Shanghai 200032, China. gyujian@citi2.net
Ni
Min
M
Xu
Jian
J
Zhang
Hongyu
H
Gao
Baomei
B
Gu
Jianren
J
Chen
Jianguo
J
Zhang
Lisheng
L
Wu
Mengchao
M
Zhen
Sushen
S
Zhu
Jingde
J
D000328:Adult; D000368:Aged; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics; D002681:China; D018899:CpG Islands / Q000235:genetics*; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics; D019175:DNA Methylation; D005260:Female; D016147:Genes, Tumor Suppressor; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D014407:Tumor Cells, Cultured; D025803:Tumor Suppressor Protein p14ARF / Q000235:genetics; D025521:Tumor Suppressor Proteins
C493966:CDKN2B protein, human; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D009363:Neoplasm Proteins; C411762:RASSF1 protein, human; D025803:Tumor Suppressor Protein p14ARF; D025521:Tumor Suppressor Proteins; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases