白血病细胞雌激素受体启动子甲基化的实验研究
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摘要
目的
不同类型的肿瘤具有特征性的DNA甲基化模式,检测基因启动子甲基化状态有助于肿瘤的诊断,还可以作为预测疗效和判断预后的分子标志。已有研究表明雌激素受体(包括ERα和ERβ亚型)基因启动子在乳腺癌、子宫内膜癌、前列腺癌等肿瘤中具有特征性的甲基化模式,而在白血病中是否也具有特征性的甲基化模式,国内还未见相关报道。本实验利用MSP方法、RT-PCR和免疫组化技术检测白血病中ERa基因启动子(ERα-A、ERα-B)和ERβ基因启动子的甲基化状态,以及ERα与ERβ的mRNA和蛋白表达的异常情况;同时观察肼苯哒嗪对ERα基因启动子去甲基化效果,以及ERα表达复活的状况。从而揭示白血病中ER基因启动子甲基化变化情况与基因表达的关系,并探讨肼苯哒嗪治疗白血病的潜在价值。
方法
1.白血病细胞株HL-60、MOLT-4、Jurkat的培养以及肼苯哒嗪对细胞株的处理。
2.应用MSP检测白血病细胞株和正常人外周血单个核细胞中ERα-A、ERα-B和ERβ基因启动子CpG岛的甲基化状态。
3.应用RT-PCR和免疫组化技术检测白血病细胞株ERα和ERβ的mRNA和蛋白表达。
4.运用MSP、RT-PCR和免疫组化技术检测肼苯哒嗪处理后白血病细胞株中ERα-A、ERα-B CpG岛甲基化状态以及ERα的mRNA和蛋白表达的变化。
结果
1.在白血病细胞HL-60、MOLT-4、Jurkat中ERα基因启动子ERα-A、ERα-B均检出甲基化,ERβ启动子为非甲基化,而正常人外周血单个核细胞中ERα与ERβ启动子区均为非甲基化。
2.在白血病细胞株HL-60、MOLT-4、Jurkat中,ERa完全失表达,而ERβ存在mRNA表达和蛋白表达。
3.肼苯哒嗪处理HL-60、MOLT-4、Jurkat后,ERα基因启动子ERα-A、ERα-B的CpG岛均发生去甲基化并检测到ERα恢复mRNA和蛋白表达。
结论
1.ER的两种亚型ERα和ERβ在白血病细胞株存在着不同的甲基化状态,而在正常人外周血单个核细胞ERα和ERβ均为非甲基化状态,提示ERα基因启动子甲基化可能是白血病的分子致病机制之一。
2.肼苯哒嗪可以使白血病细胞株ERα启动子去甲基化,同时恢复ERα基因mRNA表达和蛋白表达,说明启动子甲基化可能影响ERα基因的转录和翻译,肼苯哒嗪有望成为白血病去甲基化治疗的新药物。
3.在白血病细胞株中ERβ的表达未发现异常。
Many types of tumors have a tumor-specific methylation pattern.Methylation of gene promotor can be used to diagnose tumor and predict curative effect or duration of patient survival.There were many studys on tumor-specific methylation patter of estrogen receptor isoforms(ERa,ERβ) in several human cancers such as breast,endometrial and prostate etc, but few on leukemia in our country.This experiment was designed to detect the promotor methylation and the mRNA and protein expression of ER isoforms in leukemia cell lines by MSP(methylation specific polymerase chain reaction),RT-PCR(reverse transcription PCR) and Immunohistochemistry.On the other hand,we treated the leukemia cell lines by the demethylation reagent-Hydralazine,then detected changes in the promotor methylation and the mRNA and protein reexpression of ERα,and clarified the significance of promoter methylation ststus of ER isoforms on altered ER expression in leukemia,and investigated the potentional possibility of Hydralazine for the treatment of leukemia.
Methods:
1.Leukemia cell lines HL-60,MOLT-4 and Jurkat were cultured and treated with Hydralazine.
2.The CpG island methylation status of ERa(ERα-A,ERα-B) and ERβin Leukemia cell lines and normal human peripheral mononuclear cells were detected by MSP.
3.RT-PCR and Immunohistochemistry technology were used to analyze the expression level of ERαand ERβin HL-60,MOLT-4 and Jurkat.
4.MSP,RT-PCR and Immunohistochemistry technology were used to analyze the CpG island methylation status of ERα(A,B) and expression level of ERαin the leukemia cell lines after Hydralazine treatment.
Results:
1.MSP analysis showed that CpG islands of ERα(A,B) were methylated and ERβwas unmethylated in the three leukemia cell lines(HL-60,MOLT-4 and Jurkat).In normal peripheral mononuclear cell samples,only unmethylated bands were observed.
2.RT-PCR and Immunohistochemistry technology analysis showed that ERαwas inactived but ERβwas expressed in the three leukemia lines.
3.CpG islands of ERα(ERα-A,ERα-B)were demethylated and reexpression of mRNA and protein of ERαcould be found after Hydralazine treatment.
Conclusion
1.Aberrant promoter methylation of ERαand ERβwas found in the three leukemia lines:HL-60,MOLT-4 and Jurkat,but was not found in normal peripheral mononuclear cell samples,which indicated that DNA methylation of ER isoforms may be one of molecule mechanisms in leukemia pathogenesis.
2.After Hydralazine treatment,the CpG islands of ERαpromoter were demethylated, and the expression of mRNA and protein was restored,which demonstrated that promotor methylation may contribute to the loss of demethylated ERαexpression and Hydralazine may be noval treatment strategy for leukemia.
3.Aberrant expression of ERβwas not found in the three leukemia lines: HL-60,MOLT-4 and Jurkat.
不同类型的肿瘤具有特征性的DNA甲基化模式,检测基因启动子甲基化状态有助于肿瘤的诊断,还可以作为预测疗效和判断预后的分子标志。已有研究表明雌激素受体(包括ERα和ERβ亚型)基因启动子在乳腺癌、子宫内膜癌、前列腺癌等肿瘤中具有特征性的甲基化模式,而在白血病中是否也具有特征性的甲基化模式,国内还未见相关报道。本实验利用MSP方法、RT-PCR和免疫组化技术检测白血病中ERa基因启动子(ERα-A、ERα-B)和ERβ基因启动子的甲基化状态,以及ERα与ERβ的mRNA和蛋白表达的异常情况;同时观察肼苯哒嗪对ERα基因启动子去甲基化效果,以及ERα表达复活的状况。从而揭示白血病中ER基因启动子甲基化变化情况与基因表达的关系,并探讨肼苯哒嗪治疗白血病的潜在价值。
方法
1.白血病细胞株HL-60、MOLT-4、Jurkat的培养以及肼苯哒嗪对细胞株的处理。
2.应用MSP检测白血病细胞株和正常人外周血单个核细胞中ERα-A、ERα-B和ERβ基因启动子CpG岛的甲基化状态。
3.应用RT-PCR和免疫组化技术检测白血病细胞株ERα和ERβ的mRNA和蛋白表达。
4.运用MSP、RT-PCR和免疫组化技术检测肼苯哒嗪处理后白血病细胞株中ERα-A、ERα-B CpG岛甲基化状态以及ERα的mRNA和蛋白表达的变化。
结果
1.在白血病细胞HL-60、MOLT-4、Jurkat中ERα基因启动子ERα-A、ERα-B均检出甲基化,ERβ启动子为非甲基化,而正常人外周血单个核细胞中ERα与ERβ启动子区均为非甲基化。
2.在白血病细胞株HL-60、MOLT-4、Jurkat中,ERa完全失表达,而ERβ存在mRNA表达和蛋白表达。
3.肼苯哒嗪处理HL-60、MOLT-4、Jurkat后,ERα基因启动子ERα-A、ERα-B的CpG岛均发生去甲基化并检测到ERα恢复mRNA和蛋白表达。
结论
1.ER的两种亚型ERα和ERβ在白血病细胞株存在着不同的甲基化状态,而在正常人外周血单个核细胞ERα和ERβ均为非甲基化状态,提示ERα基因启动子甲基化可能是白血病的分子致病机制之一。
2.肼苯哒嗪可以使白血病细胞株ERα启动子去甲基化,同时恢复ERα基因mRNA表达和蛋白表达,说明启动子甲基化可能影响ERα基因的转录和翻译,肼苯哒嗪有望成为白血病去甲基化治疗的新药物。
3.在白血病细胞株中ERβ的表达未发现异常。
Many types of tumors have a tumor-specific methylation pattern.Methylation of gene promotor can be used to diagnose tumor and predict curative effect or duration of patient survival.There were many studys on tumor-specific methylation patter of estrogen receptor isoforms(ERa,ERβ) in several human cancers such as breast,endometrial and prostate etc, but few on leukemia in our country.This experiment was designed to detect the promotor methylation and the mRNA and protein expression of ER isoforms in leukemia cell lines by MSP(methylation specific polymerase chain reaction),RT-PCR(reverse transcription PCR) and Immunohistochemistry.On the other hand,we treated the leukemia cell lines by the demethylation reagent-Hydralazine,then detected changes in the promotor methylation and the mRNA and protein reexpression of ERα,and clarified the significance of promoter methylation ststus of ER isoforms on altered ER expression in leukemia,and investigated the potentional possibility of Hydralazine for the treatment of leukemia.
Methods:
1.Leukemia cell lines HL-60,MOLT-4 and Jurkat were cultured and treated with Hydralazine.
2.The CpG island methylation status of ERa(ERα-A,ERα-B) and ERβin Leukemia cell lines and normal human peripheral mononuclear cells were detected by MSP.
3.RT-PCR and Immunohistochemistry technology were used to analyze the expression level of ERαand ERβin HL-60,MOLT-4 and Jurkat.
4.MSP,RT-PCR and Immunohistochemistry technology were used to analyze the CpG island methylation status of ERα(A,B) and expression level of ERαin the leukemia cell lines after Hydralazine treatment.
Results:
1.MSP analysis showed that CpG islands of ERα(A,B) were methylated and ERβwas unmethylated in the three leukemia cell lines(HL-60,MOLT-4 and Jurkat).In normal peripheral mononuclear cell samples,only unmethylated bands were observed.
2.RT-PCR and Immunohistochemistry technology analysis showed that ERαwas inactived but ERβwas expressed in the three leukemia lines.
3.CpG islands of ERα(ERα-A,ERα-B)were demethylated and reexpression of mRNA and protein of ERαcould be found after Hydralazine treatment.
Conclusion
1.Aberrant promoter methylation of ERαand ERβwas found in the three leukemia lines:HL-60,MOLT-4 and Jurkat,but was not found in normal peripheral mononuclear cell samples,which indicated that DNA methylation of ER isoforms may be one of molecule mechanisms in leukemia pathogenesis.
2.After Hydralazine treatment,the CpG islands of ERαpromoter were demethylated, and the expression of mRNA and protein was restored,which demonstrated that promotor methylation may contribute to the loss of demethylated ERαexpression and Hydralazine may be noval treatment strategy for leukemia.
3.Aberrant expression of ERβwas not found in the three leukemia lines: HL-60,MOLT-4 and Jurkat.
引文
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