5-氮杂脱氧胞苷对子宫内膜癌细胞的生长及RASSF1A基因表达的影响

Effects and mechanisms of 5-aza-2＇-deoxycytidine on endometrial cancer cell

目的探讨DNA甲基转移酶抑制剂——5-氮杂脱氧胞苷（5-Aza—CdR）对子宫内膜癌细胞的生长及RASSF1A基因表达的影响。方法5-Aza—CdR作用后，采用锥虫蓝染色法、流式细胞仪分别检测子宫内膜癌细胞株HEC一1B细胞的生长及凋亡情况，采用RT—PCR技术、甲基化特异性PCR（MSP）技术分别检测HEC-1B细胞中RASSF1A mRNA的表达和RASSF1A基因启动子的甲基化状态。结果（1）HEC-1B细胞的生长及凋亡情况：5-Aza—CdR对HEC-1B细胞生长的抑制作用呈明显的剂量和时间依赖性（P〈0．01）；且HEC-1B细胞的凋亡率也呈明显的剂量依赖性（P〈0．01）。（2）HEC-1B细胞中RASSF1A mRNA的表达：未经5-Aza—CdR作用的HEC-1B细胞中RASSF1A mRNA表达缺失；不同浓度5-Aza—CdR（分别为0．05、0．1、1、5、10nmol／m1）作用后，HEC-1B细胞中RASSF1A mRNA表达不同程度地上升，其相对表达水平分别为0．074±0．004、0．105±0．004、0．1674-0．006、0．3344-0．005、0．484±0．007，分别与未经5-Aza—CdR作用的细胞（为0）比较，差异均有统计学意义（P〈0．01）。（3）HEC-1B细胞中RASSF1A基因启动子的甲基化状态：未经5-Aza—CdR作用的HEC-1B细胞中RASSF1A基因启动子呈高甲基化状态；加入低浓度（即0．05、0．1、1、5nmol／m1）的5-Aza—CdR后，HEC-1B细胞中RASSF1A基因启动子的甲基化水平下降，呈半甲基化状态（即同时出现甲基化和非甲基化条带）；当5-Aza—CdR浓度为10nmol／ml时，HEC一1B细胞中RASSFlA基因启动子呈非甲基化状态。结论（1）-Aza—CdR可抑制HEC-1B细胞增殖、促进HEC-1B细胞凋亡。（2）5-Aza—CdR能使HEC-1B细胞重新表达RASSF1A mRNA，能逆转RASSF1A基因启动子的高甲基化状态。

Objective To investigate the effects and mechanisms of 5-aza-2＇-deoxycytidine （5-Aza- CdR） on endometrial cancer cell. Methods In vitro experiments of 5-Aza-CdR were done using human endometrial cancer cell line HEC-1B. Evaluation of cellular proliferation and apoptosis was ascertained respectively using trypan blue exclusion and flow cytometry. RT-PCR and methylation specific PCR（MSP） was done to detect the expression of RASSF1A mRNA and methylation status of RASSF1A promoter of HEC- 1B cell line. Results （ 1 ） The status of cellular growth and apoptosis of HEC-1B cell line： the growth inhibition effects of 5-Aza-CdR on HEC-1B cell line were both concentration-dependent （ P 〈 0. 01 ） and time-dependent（P 〈0.01）, as well as the apoptosis rate of HBC-1-B cell line depended on the dose of 5- Aza-CdR obviously（P 〈 0. 01 ）. （2） The expression of RASSF1A mRNA of HEC-1B cell line： RASSF1A mRNA was expressed in HEC-1B cell after 5-Aza-CdR treatment, but it was undetectable before the treatment. In the groups with different concentration of 5-Aza-CdR （0. 05, 0. 1, 1, 5, 10 nmol/ml）, the expression of RASSF1A mRNA was respectively 0. 074 ±0. 004, 0. 105 ±0. 004, 0. 167±0. 006, 0. 334± 0. 005, 0. 484 ± 0. 007, which were remarkably different from the group without 5-Aza-CdR （ the expression of RASSF1A mRNA was 0 ; P 〈 0.01 ）. （ 3 ） The hypermethylation of RASSF1A promoter of HEC-1B cell line： the hypermethylation of RASSF1A promoter was detected in HEC-1B cell line. The status of hypermethylation was decreased after treatment with 5-Aza-CdR of 0. 05, 0. 1, 1, 5 nmol/ml, meanwhile, both methylation bands and demethylation bands were observed by methylation specific PCR. After the treatment with 5-Aza-CdR of 10 nmol/ml the hypermethylation was absent absolutely. Conclusions （ 1 ） In HEC-1B cell line, 5-Aza-CdR can inhibit cell proliferation and induce cell apopotosis. （2） 5-Aza-CdR can renew the expression of RASSF1A mRNA of HEC-1B cell line and reverse the hypermethylation of RASSF1A promoter.