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目的建立优化的染色质免疫沉淀技术(ChIP),分析乳腺癌细胞MDA-MB-231中DNA甲基转移酶3b(Dnmt3b)和组蛋白去乙酰化酶1(HDAC1)与SLC22A18基因启动子区的结合情况。方法建立并优化ChIP实验技术,运用ChIP技术检测DNA甲基转移酶抑制剂5-氮杂-2’-脱氧胞苷(5-aza-dc)和组蛋白去乙酰化酶抑制剂曲古霉素A(TSA)分别单独和联合作用于人乳腺癌细胞MDA-MB-231后,用Dnmt3b和HDAC1特异性抗体沉淀DNA,聚合酶链式反应(PCR)检测SLC22A18基因5’端特异性序列,免疫印迹法(Westernblotting)检测Dnmt3b和HDAC1蛋白的表达情况。结果获得了优化的ChIP实验条件,Dnmt3b和HDAC1抗体沉淀的染色质片段中扩增出SLC22A18基因5’端特异性序列。5-aza-dc、TSA单独用药可抑制DNMT3b、HDAC1与SLC22A18启动子区特异序列的结合,5-aza-dc和TSA联合作用可以明显抑制DNMT3b、HDAC1与SLC22A18启动子的结合;Western blotting结果表明,5-aza-dc、TSA单独及联合用药不影响DNMT3b和HDAC1蛋白的表达。结论 DNMT3b和HDAC1在MDA-MB-231细胞内结合于SLC22A18基因启动子的特异区域,参与该基因的表达调控。
OBJECTIVE: To establish an optimized chromatin immunoprecipitation assay (ChIP) to detect the expression of DNA methyltransferase 3b (Dnmt3b) and histone deacetylase 1 (HDAC1) and SLC22A18 promoter regions in breast cancer cell MDA-MB-231 Combined with the situation. Methods The ChIP experiment was established and optimized. ChIP assay was used to detect 5-aza-2-deoxycytidine (5-aza-dc) and histone deacetylase inhibitor A (TSA) alone and in combination on human breast cancer cell line MDA-MB-231, DNA was precipitated with Dnmt3b and HDAC1-specific antibodies, and the 5 ’end specific sequence of SLC22A18 gene was detected by polymerase chain reaction Western blotting was used to detect the expression of Dnmt3b and HDAC1 protein. Results The optimized ChIP experimental conditions were obtained. The 5 ’end of the SLC22A18 gene was amplified from the chromatin fragments precipitated by Dnmt3b and HDAC1 antibodies. The combination of 5-aza-dc and TSA could inhibit the binding of DNMT3b, HDAC1 and SLC22A18 promotor. The combination of 5-aza-dc and TSA could significantly inhibit the binding of DNMT3b, HDAC1 and SLC22A18 promoter. Western blotting showed that 5-aza- , 5-aza-dc, TSA alone and in combination did not affect DNMT3b and HDAC1 protein expression. Conclusions DNMT3b and HDAC1 bind to the specific region of SLC22A18 promoter in MDA-MB-231 cells, which is involved in the regulation of expression of this gene.