Transcriptional Inactivation of ERβ Gene Is Mediated by the Induction of Promoter Hypermethylation in a Rat Colonic Epithelial Cell Model

BackgroundTo construct a primary rat colonic epithelial cell model for treatment with specific methylated oligonucleotides (MOs) and to determine whether the transcriptional inactivation of ERβ mRNA is mediated by the induction of hypermethylation of the ERβ gene promoter.MethodsSuckling rat colonic epithelial cells were cultured in DMEM. Two methylated oligonucleotides complementary to the promoter regions of ERβ were synthesized and applied to the cultured cells to induce promoter hypermethylation of the ERβ gene. Methylation-specific PCR (MSP) was used to determine the methylation status of the ERβ promoter in the cultured cells. Reverse transcription–polymerase chain reaction (RT-PCR) was used to quantify the expression of ERβ mRNA after treatment with MOs.ResultsSuckling 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 ERβ 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 ERβ in the cells treated with MOs was hypermethylated compared with that of the control cells.ConclusionThe transcriptional inactivation of ERβ mRNA in rat colonic epithelial cells may be mediated by the hypermethylation of the ERβ gene promoter. Our model markedly simulates the epigenetic modification of the ERβ gene in colonic cancer cells.