[Research on promoter region methylation status of tumor repressor gene P16 in human hepatocellular carcinoma by using methylation sensitive restriction enzyme and semi-nested PCR assay].
OBJECTIVE
To construct a combination method of methylation sensitive restriction enzyme and semi-nested touch down PCR assay for studying the promoter region methylation status of P16 gene in human hepatocellular carcinoma.


METHODS
According to the sequence of CpG rich promoter region of P16 gene, three primers were designed and synthesized for semi-nested touch down PCR assay to examine the promoter region methylation status of P16 gene. 340 bp segment of this region was cloned into vector pMD18-T; the plasmid was transformed into E. coli JM109 to harvest an extended quantity, then the plasmid was treated by CpG methylase M. Sss I, the methylated plasmid was named P16Pm+. This P16Pm+ is validated by digestion of Hpa II and is employed in studying the specificity and sensitivity of this constructed method. After construction, the method was used to examine the promoter region methylation status in P16 gene of 40 DNA samples from human HCCs and three DNA samples from normal human liver tissue.


RESULTS
It was confirmed that the specificity and sensitivity of this method are solid and reliable (100 fg). It was found that 12/40 (30%) of hepatocellular carcinoma showed promoter region methylation in P16 gene whereas none (0/3) of the normal tissues was methylated in the promoter region in P16 gene.


CONCLUSIONS
Promoter region methylation in P16 gene may take part in human hepatocellular carcinogenesis. The constructed method is simple, cost-effective and is of high specificity and sensitivity, thus suggesting its potential application to detecting promoter methylation in population-based studies.
Jiang
Lei
L
Department of Biochemistry and Molecular Biology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Qin
Yang
Y
Sun
Zhi-lin
ZL
Sun
Ze-fang
ZF
Wang
Jin-hong
JH
Yang
Lu-chuan
LC
D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D019942:Genes, p16; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015203:Reproducibility of Results; D015183:Restriction Mapping / Q000191:economics / Q000379:methods*