ABSTRACT
We studied the regulatory effects of the estragen receptorβ(ERβ)gene on the downstream estrogen signal transfection pathway in colon cancer cells and the possible mechanisms involved.A human ERβ gene recombinant expression plasmid,pEGFP-C1-ERβ,was constructed and transfected into the Caco-2 colon cancer cell line,a line with low ERβ gene expression.The expression of ERβmRNA and protein was detected 72 h after transfection.RT-PCR was used to examine the expression levels of the progesterone recepror(PR)gene containing the classic estrogen response element(ERE),the C-fos oncogene containing the AP-1 site(a non-classical ER binding site),the epigenetic modifying genes,such as Dnmt1,Dnmt3a,Dnmt3b,and histone methyltransferase(HMT),and the human mismatch repair gene hMLH1.Methylation-specific PCR was used to detect the changes in the methylated sites of the CpG islands in the promoters of the ERβ,PR,and C-fos genes.The results indicated that the human ERβ gene recombinant expression plasmid pEGFP-C1-ERβ was successfully constructed and transfected into Caco-2 cells.As compared with the control group,the mRNA and protein expression of ERβ gene was increased significantly 72 h after the transfection of pEGFP-C1-ERβ into the Caco-2 cells.As compared with the control group,the mRNA expression of the PR,C-fos,Dnmt3a and Dnmt3b genes was increased significantly 72 h after the transfection of pEGFP-C1-ERβ into the Caco-2 cells,but the mRNA expression of the Dnmt1,HMT,and hMLH1 genes decreased significantly(P<0.05).As compared with the control group,different degrees of demethylation occurred in the promoters of the ERβ,progesterone receptor(PR),and C-fos oncogene 72h after the transfection of pEGFP-C1-ERβ into the Caco-2 cells.The methylation index of the estrogen signal transfection pathway in Caco-2 cells was decreased significantly following the expression restoration of ERβ gene(P<0.05).It is concluded that the restoration or up-regulation of the ERβ gene in Caco-2 cells may significantly activate the expression of the related target genes in the downstream estrogen signal transfection pathway and may result in the demethylation changes of the pathway.During the process,the expression level and activity of the epigenetic modifying genes and the human mismatch repair gene have changed simultaneously.The regulatory effect of the ERβ gene on the estrogen signal transfection pathway to a certain extent partly involves demethylation.