The impact of 5-Aza-CdR on demathylation of transcription regulation of E-cadherin gene in tumor cell line / 中国癌症杂志

ABSTRACT

Background and purpose:To explore the possibility of genetic re-expression silenced by DNA aberrant hypermethylation which is a common epigenetic modification in carcinogenesis. 5-Aza-CdR, an inhibitor of DNA methylation, was used to determine the effects of expression of tumor suppressor gene E-cadherin in tumor cell lines. Methods:Methylation specific PCR(MSP) was utilized to examine methylation status of E-cad gene on breast carcinoma cell line MDA-MB-435 before and after the treatment with 5-Aza-CdR. Immunohistochemistry(IHC) was used to test the expression of E-cad protein. Semi-quantitative RT-PCR method was used to detect the changes of E-cad mRNA.Results:1).E-cad methylation was positive(116bp) and unmethylation was negative on MDA-MB-435 cell before the treatment with 5-Aza-CdR. After being treated with 5.0umol/L 5-Aza-CdR for 3 days, methylation turned negative and unmethylation positive bands(97bp) were detected. 2).The E-cad protein expression was not detected by immunohistochemistry on MDA-MB-435 cell before the treatment, while E-cad staining was positive on the cell membrane after the treatment. 3). The E-cad mRNA failed to be amplified in cells before the treatment. After incubation at variable concentrations of 0.5 ?mol/L, 1.0 ?mol/L, 2.0 ?mol/L and 5.0 ?mol/L 5-Aza-CdR for 3 days, respectively, E-cad mRNA expression was detected on the fourth day in a dose-dependent manner. Correlation between the mRNA expression level and the agent concentration was observed.Conclusions:The demethylation agent 5-Aza-CdR can reverse the aberrant E-cad methylation status in MDA-MB-435 and re-expressed E-cad mRNA and protein.