乳腺癌细胞中PELP1基因表达与启动子区CpG岛甲基化相关性  

The correlation between expression and CpG island methylation status in promoter region of PELP1 gene in breast cancer cells

作　　者：王麟[1] 齐佳昕[2] 张金波[1] 张艳枝[1] 王鹏[3] 王晓艳[3] 梁松鹤[4] 欧芹[5] 

机构地区：[1]哈尔滨医科大学大庆校区病理生理学教研室,大庆163319 [2]哈尔滨医科大学附属第五医院心内科,大庆163319 [3]哈尔滨医科大学大庆校区生理学教研室,大庆163319 [4]哈尔滨医科大学大庆校区临床检验教研室,大庆163319 [5]佳木斯大学生化与分子生物学教研室,佳木斯154007

出　　处：《解剖科学进展》2015年第4期375-378,382,共5页Progress of Anatomical Sciences

基　　金：黑龙江省教育厅科学技术研究基金项目(No.12521237)

摘　　要：目的建立针对PELP1基因启动子区CpG岛的甲基化特异性PCR(Methylation specific PCR,MSP)检测方法,分析乳腺癌细胞中PELP1基因表达与启动区CpG岛甲基化的相关性。方法设计针对PELP1基因启动子区的MSP引物组,以甲基化和非甲基化DNA为模板验证MSP引物的特异性,建立针对PELP1基因启动子区的MSP检测方法。以DNA甲基转移酶抑制剂5'-氮杂-脱氧胞苷磷酸(5'-Aza-d C)分别处理MCF-7乳腺癌细胞、MCF-10正常乳腺导管上皮细胞,采用MSP检测PELP1基因启动子区甲基化状态变化,采用Western blot检测蛋白表达。结果针对PELP1基因启动子区设计的MSP引物组特异性良好,甲基化特异性引物仅在甲基化DNA模板获得阳性扩增条带,非甲基化引物仅在非甲基化DNA模板获得阳性条带。MCF-7乳腺癌细胞中PELP1基因启动子区呈非甲基化状态,MCF-10正常乳腺导管上皮细胞中PELP1基因启动子区呈甲基化状态,MCF-10细胞中PELP1蛋白表达水平为MCF-7细胞的1/16(P<0.05)。采用5'-Aza-dC去除MCF-10细胞PELP1基因启动子区甲基化后,PELP1蛋白表达水平上升了9.7倍(P<0.05)。结论所建立的PELP1基因启动子区MSP检测方法特异性良好,去甲基化可能是导致乳腺癌细胞中PELP1基因过表达的重要机制。Objective To establish the method of methylation specific PCR （MSP） targeting the promoter region of PEZP1 gene and analyze the correlation between the protein expression and methylation status of PELP1 in breast cancer cell. Methods MSP specific primer sets targeting CpG-island in the promoter region of PEZP1 gene was designed, the specificity of the primer sets with methylation or unmethylation specific DrqA templet was verified. MCF-7 breast cancer cell line and MCF-IO normal breast duct epithelia cell line were treated with 5＇ -Aza-dC, the methylation status and protein expression of PEZFlwere examined respectively by MSP and Western blot. Results MSP primer sets showed good specificity with only positive M primer pair amplificated band in methylated templet as well as U primer pair amplificated band in unmethylation templet. CpG-island in PEZPI promoter region showed hypomehtylation in MCF-7 and hypermethylaiton in MCF-10, the level of PELP1 protein in MCF-10 was 1/16 fold of that in MCF-7. MCF-10 treated with 5＇ -Aza-dC induced demethylaiton of CpG-island in PEZPlpromoter region and increased level of PELP1 protein to 9.7 fold. Conclusion MSP method showed good specificity, demethylation of promoter region of PELFI gene may be an important mechanism of overexpression of PELP1 in breast cancer cells.

关 键 词：乳腺癌 脯氨酸-谷氨酸-亮氨酸富集蛋白1 表观遗传学 甲基化特异性PCR 

分 类 号：R739.9[医药卫生—肿瘤]