Qian Hu1, Li Yu, Qin-ping Liao. 1. Department of Gynecology and Obstetrics, Peking University First Hospital, Beijing, China.
Abstract
OBJECTIVE: To investigate the regulation of DNA methylation on the expression of MMP-2 gene, and the effect of 5-aza-2'-deoxycytidine (5-aza-CdR) on invasiveness of endometrial cancer cells. METHODS: Human endometrial cancer cell lines Ishikawa and HEC-1A were treated with 5-aza-CdR. After genomic DNA was extracted from the cells and treated with bisulfate sodium, clone sequencing was used to detect methylation of the matrix metalloproteinases-2 (MMP-2) promoter region. Ishikawa and HEC-1A cells were treated with different concentrations of 5-aza-CdR, and the effect on the invasion and metastasis to the endometrial cancer (EC) cells were detected by Transwell invasion chamber. The MMP-2 mRNA expression was detected by real-time quantitative PCR. RESULTS: Although the MMP-2 promoter showed global hypomethylation status, the methylation of CG dinucleotide sites located on the transcription factor binding sites had high positive frequencies, which were 80% and 70% in HEC-1A cells and 60% and 40% in Ishikawa cells, respectively. The methylation frequencies reduced to 20% and 10% in HEC-1A cells by demethylating agent 5-aza-CdR and the CG sites in Ishikawa cells were totally demethylated. Moreover, after 72 h of treatment with different concentrations of 5-aza-CdR, the expression of the MMP-2 mRNA and transmembrane number of both Ishikawa and HEC-1A cells were significantly upregulated compared with the control group (P<0.05). CONCLUSION: Methylation of CG dinucleotide sites of the MMP-2 promotor region may be associated with the regulation of gene transcription. The 5-aza-CdR can promote the invasion and metastasis of endometrial cancer cells in vitro by upregulating the MMP-2 mRNA expression.
OBJECTIVE: To investigate the regulation of DNA methylation on the expression of MMP-2 gene, and the effect of 5-aza-2'-deoxycytidine (5-aza-CdR) on invasiveness of endometrial cancer cells. METHODS:Humanendometrial cancer cell lines Ishikawa and HEC-1A were treated with 5-aza-CdR. After genomic DNA was extracted from the cells and treated with bisulfate sodium, clone sequencing was used to detect methylation of the matrix metalloproteinases-2 (MMP-2) promoter region. Ishikawa and HEC-1A cells were treated with different concentrations of 5-aza-CdR, and the effect on the invasion and metastasis to the endometrial cancer (EC) cells were detected by Transwell invasion chamber. The MMP-2 mRNA expression was detected by real-time quantitative PCR. RESULTS: Although the MMP-2 promoter showed global hypomethylation status, the methylation of CG dinucleotide sites located on the transcription factor binding sites had high positive frequencies, which were 80% and 70% in HEC-1A cells and 60% and 40% in Ishikawa cells, respectively. The methylation frequencies reduced to 20% and 10% in HEC-1A cells by demethylating agent 5-aza-CdR and the CG sites in Ishikawa cells were totally demethylated. Moreover, after 72 h of treatment with different concentrations of 5-aza-CdR, the expression of the MMP-2 mRNA and transmembrane number of both Ishikawa and HEC-1A cells were significantly upregulated compared with the control group (P<0.05). CONCLUSION: Methylation of CG dinucleotide sites of the MMP-2 promotor region may be associated with the regulation of gene transcription. The 5-aza-CdR can promote the invasion and metastasis of endometrial cancer cells in vitro by upregulating the MMP-2 mRNA expression.