Epigenetic Regulation of the ERβ Gene on the Estrogen Signal Transfection Pathway in Colon Cancer Cells / 华中科技大学学报（医学）（英德文版）

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.

Induction of Apoptosis of Human Colon Cancer Cells by siRNA Recombinant Expression Vector Targeting Survivin Gene / 华中科技大学学报（医学）（英德文版）

This study constructed siRNA recombinant expression vector targeting survivin gene and observe the apoptosis induction effect of it in human colon cancer cells,siRNA recombinant expres-sion vector targeting survivin gene was constructed and transfected into human colon cancer cells.The effect of siRNA recombinant expression vector was detected by RT-PCR,Western blot,MTT re-duction assay and flow cytometry.It was confirmed by restriction endonuclease and sequence analy-sis that siRNA recombinant expression vector targeting survivin gene was constructed successfully.Inhibition rate of survivin siRNA at mRNA and protein levels was 36.33% and 44.65% respectively.Growth of cancer cells was inhibited and the apoptosis rate was (17.24±2.13)%.The siRNA recom-binant expression vector targeting survivin gene has been constructed successfully.It not only can in-hibit the expression of survivin gene,but also can induce apoptosis in human colon cancer cells re-markably.