Analysis to DNA methylation of the promoter of human telomerase reverse transcriptase (hTERT) in gastric cell lines / 解放军医学杂志

ABSTRACT

Objective To examine and analyze the methylation status of the promoter region of human telomerase reverse transcriptase(hTERT)in gastric cell lines.Methods Telomerase activity was detected with telomeric repeat amplification protocol(TRAP).Expression of hTERT was observed by immunofluorescence.The target sequence of hTERT CpG island(-692~-403bp)was selected by database search.The methylation status of hTERT CpG island was examined with bisulfite sequencing PCR(BSP)as followingGenomic DNAs were modified by sodium bisulfite and amplified by PCR with primers that contained no CpG sites.Direct sequencing was performed for each case.Results Both TRAP and immunofluorescence analysis showed that high levels of telomerase and hTERT were expressed in SGC-7901 and BGC-823 cells.The sequences(-634~-403bp)of hTERT gene promoter,which were sequenced successfully,were homologous to the sequences before sodium bisulfite modification.25 CpGs in BGC-823 cell line and 26 CpGs in SGC-7901 cell line were observed to be methylated in all 26 CpGs of this part.Only one repressive transcription factor,which called myeloid-specific zinc finger protein 2(MZF-2),was found to bind this region which possessed two special binding motifs.Such a kind of binding showed potential importance for methylation-mediated derepression of hTERT transcription.Conclusion The hypermethylation may change the structure of DNA and result in abortive binding between MZF-2 and its motif,which might be one of the reasons for up-regulation of hTERT transcription and telomerase activity.