去甲斑蝥素对食管癌细胞株Eca-109中hTERT的表达和PTPRO基因启动子甲基化影响的实验研究

Hypermethylation of PTPRO and expression of hT ERT in human esophageal cancer cell line Eca-109 treated with norcantharidin

目的探索去甲斑蝥素(NCTD)诱导食管癌细胞Eca-109对人端粒酶逆转录酶(hT ERT)表达和受体O型蛋白酪氨酸磷酸酶(PTPRO)基因启动子甲基化的影响。方法 MTT比色法检测不同浓度(0、1、2、4、8、16μg/ml)NCTD及不同作用时间(12、24 h)对食管癌细胞Eca-109增殖的影响;TUNEL法配合激光共聚焦扫描显微镜检测NCTD+Eca-109组(实验组)和Eca-109组(对照组)细胞凋亡情况;甲基化特异性PCR(MSP)检测实验组和对照组中PTPRO基因启动子的甲基化状态,Western blotting检测实验组和对照组hT ERT蛋白的表达。结果 MTT检测显示,NCTD可抑制Eca-109细胞增殖,呈时间和浓度依赖性,不同作用时间和浓度的细胞增殖抑制率的差异有统计学意义(P<0.05);TUNEL法结合激光共聚焦扫描显微镜观察显示,实验组细胞内部结构发生剧烈的变化,较对照组细胞核染色质致密浓染,核形缩小;MSP检测显示,对照组PTPRO基因启动子发生明显甲基化;Western blotting检测显示,实验组hT ERT蛋白表达较对照组明显降低(P<0.05)。结论NCTD对食管癌细胞Eca-109有细胞毒性,能够抑制细胞生长,其机制可能与下调hT ERT蛋白表达和PTPRO基因启动子去甲基化有关。

Objective To investigate the expression of human telomerase reverse transcriptase（ hT ERT） and hypermethylation of proteintyrosine phosphatase receptor（ PTPRO） in human esophageal cancer cell line Eca-109 treated with norcantharidin（ NCTD）. Methods Eca-109 cells were treated with different concentrations of NCTD（ 1,2,4,8,16 μg/ml） and the proliferation was determined by MTT assay at 12 h and 24 h. Cell apoptosis of NCTD ＋ Eca-109 group（ experimental group） and Eca-109 group（ control group） were detected by TUNEL and laser scanning confocal microscope（ LSCM）. Methylation-specific PCR（ MSP） was used to detect the PTPRO methylation status in both experimental and control groups. hT ERT protein was tested using Western blotting. Results MTT method showed that NCTD could increase the proliferation inhibitory rate of Eca-109 cells in a dose and time depended manner. The proliferation inhibitory rates of Eca-109 cells were statistically different among different concentrations of NCTD,as wall as different exposure time. Distinct morphological changes of apoptosis were found in experimental group tested by TUNEL and LSCM. PTPRO methylation status was found in control group by MSP test. The expression of hT ERT protein was reduced in experimental group compared with control group（ P〈 0. 05）. Conclusion NCTD can significantly inhibit the growth of esophageal cancer cell line Eca-109,which is associated with the down-regulation of hT ERT and PTPRO methylation status.