Mapping the methylation pattern by bisulfite genomic sequencing of the E-cadherin promoter CpG island in nasopharyngeal carcinoma.
BACKGROUND
It is hypothesised that hypermethylation of the CpG island in the promoter region in several tumour suppressor genes contributes one of the mechanisms for silencing or down-regulating the expression of the tumour suppressor genes. One of the putative tumour suppressor genes, the E-cadherin gene, has shown down-regulation in nasopharyngeal carcinoma (NPC). The aim of this study was to explore the methylation profiles of the E-cadherin promoter region in NPC patients.


METHODS
Fifteen NPC paraffin-embedded tissue sections were included in the study accompanied by two cell lines, MCF7 and MDA435, as controls. The technique of bisulfite genomic sequencing was used to determine the methylation patterns of the 29 CpG dinucleotides along the E-cadherin promoter region. DNAs were extracted from the samples, and then were subject to bisulfite conversion. Nested polymerase chain reactions were performed on the modified DNAs, the sequences of which were determined by direct sequencing.


RESULTS
A pilot study ascertained the correlation between reduced E-cadherin expression and hypermethylation of the E-cadherin promoter region from the E-cadherin (+) MCF7 and the E-cadherin (-) MDA435 cell lines. The experimental study revealed that, among 10 NPC cases in which DNA could be successfully extracted, 6 (60%) displayed methylation patterns at two or more CpG sites (range: 2-11, 6.8-37.9%). Five of them (83.3%) were comprised of at least 2 transcription enhancing factor sites. Two of the 4 cases (50%) with methylated 4th CpG site (HpaII site) and 3 of the 4 cases (75%) with methylated 11th CpG site (GC region) had grades 3 or 4 level of methylation.


CONCLUSIONS
The data indicated that hypermethylation of the E-cadherin promoter region occurred in a large proportion of NPC patients, and frequent methylation of specific CpG sites associated with transcription enhancing elements suggested that this phenomenon may contribute part of the mechanisms for silencing or down-regulating E-cadherin gene expression in NPC.
Kao
Ruey Ho
RH
Department of Hematology/Oncology, Tzu Chi General Hospital, 707, Section 3, Chung Yang Road, Hualien, 970, Taiwan. rueykao@mail.tcu.edu.tw
Huang
Li Chi
LC
Hsu
Yung Hisang
YH
D001483:Base Sequence; D015820:Cadherins / Q000235:genetics*; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D008969:Molecular Sequence Data; D009303:Nasopharyngeal Neoplasms / Q000235:genetics*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods; D013447:Sulfites
D015820:Cadherins; D013447:Sulfites; C042345:hydrogen sulfite