Epigenetic Modulation On Estrogen Signaling By Aging Methylation Of ERβ Gene In Colon Cancer Cells

Objectives:It is indicated that estrogen receptor (ER) gene may play an important role in the process of modifications of histone tails and nucleosome remodeling by recruiting transcription factors such as Histones acetyltransferases (HAT) and Histone methyltransferase(HMT),which suggests that ERβgene can also mediate epigenetic regulation on its downstream targeted genes and consequently participates in carcinogenesis except for the classical signaling pathway via estrogen reaction element(ERE).Then how does it perform this process in colon cancer?Since promoter hypermethylation could result in transcription inactivation and transcription inactivation could mediate epigenetic regulation on its downstream targeted genes,then is there a cause and effect relationship between promoter hypermethylation of ERβgene and its epigenetic regulation?In other words, Does whether aging hypermethylation of ERβpromoter trigger the epigenetic regulation on its downstream targeted genes or not? This research is just executed to try to answer these questions and to know about pathologic mechanism of ERβgene aging hypermethylation and further the pathogenesis of the female ascending colon caner.Methods:Promoter CpG islands methylation modes of ERβ, progesterone receptor (PR) and c-fos genes were determined by methylated specific PCR(MSP) and the mRNA levels of ERβ,DNA Methyltransferase(Dnmt) 1, Dnmt3a,Dnmt3b, Histone Methyltransferase(HMT) and hMLH1 genes were detected by RT-PCR in HT-29,Lovo and Caco-2 colon cancer cells,separately. The expression level of ERβgene was artificially down-regulated using small interfering RNA(siRNA) and up-regulated by treatment with estradiol or ERβeukaryotic expression vector,separately, to determine the modulating action on estrogen signaling by ERβgene and the possible mechanism in Caco-2 cells.For the first time, we have established a new functional primary culture cell model by inducement of ERβgene promoter aging hypermethylation in Sprague Dawley rat normal colonic epithelial cells using a novel methylated oligonucleotides(MOs).Suckling rat colonic epithelial cells were primary cultured in DMEM media. Two MOs complementary to promoter regions of ERβwere synthesized and used on the cultured cells to induce promoter hypermethylation of ERβgene.MSP method was used to determine the methylation status of ERβpromoter and RT-PCR was used to detect the expression abundances of ERβmRNA in the cultured cells after MOs treatment.To study the epigenetic regulation on downstream target genes by aging hypermethylation of ERβgene,the expression levels of PR,c-fos,hMLH1,and epigenetic enzymatic spectrum genes,such as Dnmt1,Dnmt3a,Dnmt3b and HMT,were detected by RT-PCR in the cell model 48hours after MOs treatment.Results:1.The ERβand PR genes promoter were hypermethylated in HT-29,Lovo and Caco-2 colon cancer cells(P<0.01), whereas the c-fos gene promoter which contained the AP-1 site, partly showed a methylated CpG islands in all the three cancer cells(P<0.05).2.It was indicated that significant overexpression of Dnmt3b was seen in the human colon cancer cells while Dnmt1, HMT and hMLH1 genes were only modestly overexpressed.Dnmt3a and ERβgenes showed a very low expression level in all the three cancer cells. A significant discrimination of Dnmt3b mRNA expression levels was found between HT-29,Lovo and Caco-2 cells because Dnmt3b was present at high level in HT-29 and Lovo cell lines but declined drastically in Caco-2 cells.3.siRNA targeted ERβgene in Caco-2 cells could not change promoter hypermethylation modes of ERβ,PR and c-fos genes as well as the expression levels of Dnmt1,Dnmt3a,Dnmt3b and HMT genes.It seemed to have no effect on estrogen signaling pathway by down-regulating ERβgene expression in Caco-2 cells.4.Caco-2 cells were treated with 10-6mol/L or 10-8mol/L estradiol for 2 weeks to induce activation of ERβgene,but it failed.ERβeukaryotic expression vector was constructed successfully and then transfected into Caco-2 cells to reactivate ERβexpression. The mRNA levels of PR,c-fos,Dnmt3a and Dnmt3b gene were up-regulated in Caco-2 cells transfected with the recombinant compared with the control(P<0.01). The mRNA levels of Dnmt1,HMT and hMLH1 gene were down-regulated in Caco-2 cells transfected with the recombinant(P<0.01). At the same time,ERβ,PR and c-fos genes displayed an incomplete demethylating modifications in Caco-2 cells transfected with the recombinant.5.Suckling rat colonic epithelium cells was successfully cultured in vitro.Identification by immunohistochemical staining showed that more than 95% of the cultured cells were epithelial. The methylated and unmethylated oligonucleotides(UMOs) we designed and synthesized were transfected into the colonic epithelium cells successfully and assembled in the nucleolus of cells which had a better proliferative activity.RT-PCR demonstrated that expression of ERβmRNA was significantly suppressed in the cells treated with MOs, whereas in the cells treated with UMOs as control was not.MSP analysis showed that ERβgene promoter in the cells treated with MOs was hypermethylated compared with control. 6.Further study indicated that expressions of downstream targeted genes of estrogen signaling pathway were repressed simultaneously when ERβmRNA was significantly suppressed in the cells treated with MOs.What's more,the activity of epigenetic enzymatic spectrum genes(Dnmt1,Dnmt3a,Dnmt3b and HMT) has changed at the same time.Conclusions:The present results suggested that except for the classical signaling pathway via estrogen reaction element,aging hypermethylation of ERβgene promoter can also remarkably mediate epigenetic regulation on its downstream targeted genes and consequently result in turbulence of this signaling pathway,which may benefit the effort to seek for the pathogenesis of the female ascending colon caner.Transcriptional inactivation of ERβgene in colonic epithelium cells may be mediated by promoter hypermethylation of ERβgene.Aging hypermethylation of ERβand its modulation on downstream targeted genes may play a significant role in carcinogenesis in colon cancer.Detection of the abnormal hypermethylation mode of ERβgene promoter will contribute to early diagnosis and prognosis evaluation of the female ascending coloh cancer.