【作者】 刘剑；

【导师】 王留兴； 陆士新；

【作者基本信息】 郑州大学 ， 肿瘤学， 2007， 硕士

【摘要】 ER-α基因定位于染色体6q24～27上，其表达的ER-α蛋白常作为一个重要分子标志物用于评估乳腺癌治疗的激素依赖性和预测内分泌治疗效果；ER-α基因失表达（ER-α阴性）的乳腺癌患者对内分泌治疗不敏感，预后差。据文献报道ER-α基因表达沉默与基因重排、突变、缺失等无关，可能与其启动子区CpG岛异常甲基化有关。因此，针对失表达ER-α基因的去甲基化治疗成为雌激素受体阴性乳腺癌治疗的一条新思路。本课题为此进行该方向的实验研究。本实验选用国内最早报道原用于诱导治疗的化学物质（As₂O₃）作为去甲基实验药物，开展对ER-α基因高甲基化的乳腺癌细胞系MDA-MB-231细胞去甲基作用机制的实验研究，探讨As₂O₃对乳腺癌细胞MDA-MB-231的去甲基化效果及其同基因表达的关系，旨在研究As₂O₃与乳腺癌细胞DNA甲基化的关系及其去甲基化治疗的可能机制，并为As₂O₃作为去甲基化药物提供实验依据。材料与方法：（1）选用雌激素受体（ER）阴性人乳腺癌细胞株MDA-MB-231常规培养，将对数生长期的MDA-MB-231细胞调整细胞浓度为5×10⁵/mL，换含As₂O₃的L-15培养液，至As₂O₃终浓度分别为0.5μmol/L、2.0μmol/L、4.0μmol/L，连续培养72小时。以不加药同期培养的MDA-MB-231细胞作为阴性对照。以同期培养在RPMI-1640环境中的雌激素受体（ER）阳性人乳腺癌细胞株MCF-7为阳性对照。（2）采用REP-PCR方法检测ER-α基因外显子Ⅰ（含过度甲基化的CpG岛）的表达。（3）采用Western Blot技术检测经不同浓度As₂O₃处理后的MDA-MB-231细胞ER-α蛋白的表达情况。（4）MTT技术检测不同浓度As₂O₃处理后的MDA-MB-231细胞，再经他莫昔芬处理，各组细胞增殖抑制率的差异。（5）实验数据经SPSS11.0统计软件处理，采用单因素方差分析，以α=0.05为差异显著性标准。结果：（1）REP-PCR法检测未经As₂O₃处理的MDA-MB-231细胞在经NotI酶过量消化前后，均可扩出清晰的外显子Ⅰ片段，提示ER-α基因甲基化阳性；一定浓度（2.0μmol/L和4.0μmol/L）的As₂O₃作用72小时后，再次酶切后扩增未见目的条带，提示ER-α基因甲基化阴性；以MCF-7细胞为阳性对照，经NotI酶过量消化后也未扩增出ER-α基因外显子Ⅰ片段。（2）经Western Blot检测未经As₂O₃处理的MDA-MB-231细胞未显示ER-α蛋白带，0.5μmol/L As₂O₃处理的MDA-MB-231细胞也未显示ER-α蛋白带，但经2.0μmol/L和4.0μmol/L As₂O₃处理的MDA-MB-231细胞可见ER-α蛋白带。（3）MTT法检测他莫昔芬作用于三氧化二砷处理过的MDA-MB-231细胞后，细胞增殖明显受到抑制。浓度2.0μmol/L左右的As₂O₃应当是MDA-MB-231细胞株去甲基化体外实验的较适合剂量，虽然As₂O₃处理后MA-MB-231细胞对TAM的敏感性不及MCF-7细胞，但明显优于未经处理的MA-MB-231细胞，提示TAM对As₂O₃处理过的MDA-MB-231细胞有明显的细胞毒作用。结论：1．一定浓度的As₂O₃能够使MDA-MB-231细胞系ER-α基因中的CpG岛发生去甲基化。2．一定浓度的As₂O₃可诱导MDA-MB-231细胞系ER-α蛋白表达。3．As₂O₃诱导MDA-MB-231细胞表达的ER-α是有功能的。4．As₂O₃通过去甲基化作用使雌激素受体阴性的乳腺癌细胞表达有功能的雌激素受体，从而为雌激素受体阴性的乳腺癌患者接受内分泌治疗提供理论依据。更多还原

【Abstract】 Objective: ER-αgene is located on the chromosome 6q24～27.The proteinum expressed by ER-αgene is usually an important molecule marker to evaluate hormonal dependence and predict the effect of endocrine therapy on breast cancer. The patients of breast cancer （ER-αnegative） are not sensitive to endocrine therapy and more malignant poor prognosis.Current reasearches report the mechanism of ER-αgene expression silencing is not due to generearrangement, mutation , absence and so on ,but probably associated with the CpG island methylation in the core Promotor of ER-αgene. Thus ,demethylation therapy aimed at ER-αgene becomes a new therapeutic strategy for breast cancer.Now we choose arsenic trioxide which used as inductive treatment drug as experiment drug of demethylation. The purpose is to investigate the demethylating effects on ER-αgene in MDA-MB-231 cell and the relations between demethylation and gene expression, to explore the correlation between arsenic trioxide and DNA methylaion and clarify the possible mechanisms of demethylating treatment, also to provide experimental basis for arsenic trioxide as methylation inhibitor.Methods and materials:（1） Human breast cell line MDA-MB-231 were growing at 37℃and 5% CO₂ MDA-MB-231 were plated at densities of 5×10⁵ cells/100-mm dish and were treated with L-15 containing As₂O₃ 0.5μmol/L、2.0μmol/L、4.0μmol/L respectively. The media containing the drug was maintained during the 3 days of treatment. MDA-MB-231 without being treated with containing As₂O₃ were taken as negative control . Human breast cell line MCF-7(（ER negative） were taken as positive control.（2） The MDA-MB-231 cell line can express or not exon I fragment of the estrogen receptor gene （containing the CPG island of hypermethylation） which are determined by REP-PCR technique.（3） Western Blot was performed for ER-αprotein after the MDA-MB-231 cell line in which the estrogen receptor gene is silenced due to hypermethylation was treated with different concentration of As₂O₃（4） The tamoxifen induced growth inhibition of MDA-MB-231 cell after was treated with different concentration of As₂O₃ by MTT method.（5） Experimental data are disposed by SPSS11.0 statistics soft ware,deploying one-way analysis of variance, significance of difference standard （α=0.05）. Results:（1） ER-αgenes of MDA-MB-231 cells of no drugs can clearly amplificate exon I fragment before or behind excessive digestion with restricted enzyme NotI. It hints ER-αgenes containing the hypermethylation part; But ER-αgenes can’t amplificate exon I fragment treated with 2.0μmol/L and 4.0umol/L As₂O₃ for three days and excessive digested with restricted enzyme Not I, it hints negative for hypermethylation; Taking MCF-7 cells as positive control, it can’t amplificate exon I fragment too behind excessive digestion with restricted enzyme NotI.（2） Detectable expression of ER-αprotein was found in the MDA-MB-231 cells treated with 0.5umol/L As₂O₃ determined with Western blot technique But detectable expression of ER-αprotein was found in the MDA-MB-231 cells treated with 1 .0μmol/L、2.0μmol/L and 4.0μmol/L As₂O₃ determined with Western blot technique;（3） Tamoxifen could inhibit the growth of the MDA-MB-231 cells treated with As₂O₃ efficiently through MTT methods, arsenic trioxide（2.0μmol/L） should be the better proper dose in vitro experiment of demethylation of the MDA-MB-231 cells,although they are less sensitive to TAM than MCF-7 cells and obviouslay better than the MDA-MB-231 cells without being disposed by arsenic trioxide. so TAM has obviously toxic action of the MDA-MB-231 cells treated by arsenic trioxide.Conclusion:（1） At definite concentration, As₂O₃ could make the CPG island of ER-αgene shows demethylation in human breast cell line MDA-MB-231 .（2） At definite Concentration, As₂O₃ could induce ER-αprotein re-expression in human breast cell line MDA-MB-231 through DNA demethylation. （3） The re-expressed genes are functional in human breast cell line MDA-MB-231 through DNA demethylation.（4） As₂O₃ could induce ER-αfunctional gene re-expression in human breast cell line MDA-MB-231 through DNA demethylation. Thus provide an evidence in theory for breast cancer patients （ER negative） to take endocrine therapy.更多还原