Transcriptional inactivation of ERbeta gene is mediated by the induction of promoter hypermethylation in a rat colonic epithelial cell model.
BACKGROUND
To construct a primary rat colonic epithelial cell model for treatment with specific methylated oligonucleotides (MOs) and to determine whether the transcriptional inactivation of ERbeta mRNA is mediated by the induction of hypermethylation of the ERbeta gene promoter.


METHODS
Suckling rat colonic epithelial cells were cultured in DMEM. Two methylated oligonucleotides complementary to the promoter regions of ERbeta were synthesized and applied to the cultured cells to induce promoter hypermethylation of the ERbeta gene. Methylation-specific PCR (MSP) was used to determine the methylation status of the ERbeta promoter in the cultured cells. Reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify the expression of ERbeta mRNA after treatment with MOs.


RESULTS
Suckling rat colonic epithelial cells were successfully cultured in vitro. The MOs and unmethylated oligonucleotides (UMOs) we designed, synthesized, successfully transfected into the colonic epithelial cells, and assembled in the nuclei of the cells, which had extremely elevated proliferative activity. RT-PCR demonstrated that the expression of ERbeta mRNA was significantly suppressed in the cells treated with MOs, whereas its expression in the control cells treated with UMOs was not. MSP analysis showed that the promoter of ERbeta in the cells treated with MOs was hypermethylated compared with that of the control cells.


CONCLUSIONS
The transcriptional inactivation of ERbeta mRNA in rat colonic epithelial cells may be mediated by the hypermethylation of the ERbeta gene promoter. Our model markedly simulates the epigenetic modification of the ERbeta gene in colonic cancer cells.
Zhai
Rong Lin
RL
Department of General Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. zhaironglin@yahoo.com.cn
Wang
Guo Bin
GB
Cai
Kai Lin
KL
Tao
Kai Xiong
KX
Xu
Fei
F
Zhang
Wan Li
WL
Wang
Zhi Yong
ZY
D000818:Animals; D002478:Cells, Cultured; D003106:Colon / Q000187:drug effects / Q000378:metabolism*; D003110:Colonic Neoplasms / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects*; D044127:Epigenesis, Genetic / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D047629:Estrogen Receptor beta / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D023421:Models, Animal; D009841:Oligonucleotides / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014158:Transcription, Genetic / Q000187:drug effects*
D047629:Estrogen Receptor beta; D009841:Oligonucleotides; D012333:RNA, Messenger