雌激素通过调节三叶因子3基因启动子甲基化水平促进甲状腺癌细胞增殖的研究

Estrogen promotes the proliferation of thyroid carcinoma cells by regulating the methylation level of trefoil factor 3 gene promoter

目的 探讨17β-雌二醇对甲状腺癌细胞增殖的影响,及其与三叶因子3(TFF3)基因启动子甲基化水平的关系。方法 将甲状腺癌TPC-1、BCPAP、FTC-133细胞分为4组:空白组(未加任何处理)、对照组(含浓度<0.05%二甲基亚砜的培养基)和低、高剂量实验组(1和100 nmol·L^(-1) 17β-雌二醇)。用甲基化特异性聚合酶链反应分析17β-雌二醇处理前后3株细胞TFF3基因启动子区甲基化水平,用生长曲线检测17β-雌二醇对3株细胞增殖的影响。结果 TFF3基因启动子甲基化水平随17β-雌二醇浓度的升高而降低。在TPC-1细胞中,低、高剂量实验组的TFF3基因启动子区甲基化百分比分别从空白组和对照组的(53.34±0.62)%,(54.62±3.11)%降至(37.12±1.19)%和(27.84±0.73)%,BCPAP细胞的TFF3基因启动子区甲基化百分比从空白组和对照组的(56.04±0.93)%,(58.08±0.89)%降至(50.33±0.55)%和(26.36±0.74)%,FTC-133细胞的TFF3基因启动子区甲基化百分从空白组和对照组的(73.28±0.36)%,(71.64±0.54)%降至(61.33±0.56)%和(51.28±0.62)%。在3种细胞中,低、高剂量实验组的上述指标与对照组相比,差异均有统计学意义(P<0.05,P<0.01)。生长曲线结果显示,17β-雌二醇能促进甲状腺癌细胞增殖。对照组在第5天时TPC-1,BCPAP和FTC-133细胞的汇合度分别为(50.04±2.83)%,(30.02±4.97)%和(25.09±2.94)%,高剂量实验组在第5天时TPC-1,BCPAP和FTC-133细胞的汇合度分别上升到(100.00±2.16)%,(90.08±3.56)%和(40.03±2.93)%,高剂量实验组的上述指标与对照组相比,差异均有统计学意义(均P<0.01)。结论 17β-雌二醇能通过降低TFF3基因启动子区CPG位点的DNA甲基化水平,促进癌细胞增殖。

Objective To investigate the effect of 17β-estradiol on proliferation of thyroid cancer (TC) cells,and to explore its relationship with the methylation levels of trefoil family factor 3 (TFF3) promoter in different TC cell lines.Methods The 3 types of TC cells,TPC-1,BCPAP and FTC-133 were divided into 4 groups:blank group (without any treatment),control group (media containing<0.05%dimethylsulfoxide),experimental-L group (1 nmol·L^(-1)) and experimental-H group (100 nmol·L^(-1)).The methylation levels of TFF3 gene promoter region of TC cells before and after 17β-estradiol treatment were analysed by methylation-specific polymerase chain reaction.Growth curve experiment was used to detect the effect of 17β-estradiol on the proliferation of three types of TC cells.Results The methylation level of the TFF3 gene promoter decreased with the increase of 17β-estradiol concentration.In TPC-1 cells,the methylation percentage of TFF3 gene promoter region in blank and control groups were (53.34±0.62)%and (54.62±3.11)%,which in experimental-Land experimental-H groups decreased to (37.12±1.19)%and (27.84±0.73)%,respectively.In BCPAP cells,the methylation percentage of TFF3 gene promoter region in blank and control groups were (56.04±0.93)%and(58.08±0.89)%,which in experimental-L and experimental-H groups decreased to (50.33±0.55)%and(26.36±0.74)%,respectively.In FTC-133 cells,the methylation percentage of TFF3 gene promoter region in blank and control groups were (73.28±0.36)%and (71.64±0.54)%,which in experimental-L and experimental-H groups decreased to (61.33±0.56)%and (51.28±0.62)%,respectively.Among the three cells,the differences of above indexes were statistically significant between the experimental-L,-H groups and control group (P<0.05,P<0.01).The results of growth curve indicated that 17β-estradiol could stimulate the proliferation of thyroid cancer cells.The confluences of TPC-1,BCPAP and FTC-133 cells at the 5^(th)day were(50.04±2.83)%,(30.02±4.97)%,(25.09±2.94)%in blank group,but in group experimental-H,these values rised to (100.00±2.16)%,(90.08±3.56)%,(40.03±2.93)%.Also,there were statistically significant differences between experimental-H group with blank group (all P<0.01).Conclusion 17β-estradiol may promote TC cell proliferation by reducing the DNA methylation level of CPG sites in the promoter region of TFF3 gene.