摘要
目的:观察常染色质组蛋白甲基转移酶1(euchromatic histone methyltransferase1,EHMT1)与金属硫蛋白1h(metallothionein1h,MT1h)的相互作用及定位,探讨其对组蛋白H3K9甲基化和肺癌细胞生物学行为的影响及可能的作用机制。方法:将pGBKT7-MT1h质粒与pGADT7-EHMT1质粒共同转化至酵母菌株AH109,进行酵母双杂交实验。将pcDNA4/TO/myc-hisA/MT1h与pcDNA6/TR质粒共转染肺癌细胞株A549和A2,并用四环素诱导MT1h表达后,采用免疫共沉淀和激光共聚焦法检测MT1h、标签蛋白c-myc和EHMT1蛋白的表达及定位;运用组蛋白H3K9甲基转移酶试剂盒检测甲基化转移酶活性,Western印迹法检测三甲基化的组蛋白H3K9表达;FCM和克隆形成实验检测MT1h表达对细胞增殖的影响;划痕实验和Borden小室法检测MT1h对细胞迁移的影响。结果:实验证实MT1h与EHMT1存在直接作用,且二者共定位于细胞核。诱导MT1h表达后,组蛋白甲基化转移酶活性升高,并且MT1h与组蛋白赖氨酸H3K9三甲基化密切相关。功能分析显示,MT1h稳定转染的肺癌细胞多处于G0期,细胞运动和迁移能力降低。结论:MT1h与EHMT1在细胞核内直接作用,可能通过调节组蛋白H3K9甲基化来发挥抑癌基因的作用。
Objective： To confirm the direct binding of euchromatic histone methyltransferase 1 （EHMT1） and metallothionein lh （MTlh）, and to explore the mechanism and impact on histone H3K9 methylation and lung cancer cells. Methods： pGBKT7-MTlh and pGADT7-EHMT1 plasmids were co-transfected into yeast AH109. pcDNA4/TO/myc-hisA/MTlh and pcDNA6/TR plasmids were co-transfected into human lung cancer cell lines A549 and A2. The expression of MTlh was induced by tetracycline. The expressions of MTlh, tag c-myc and EHMT1 proteins were detected by yeast two-hybrid test, co-immunoprecipitation assay and GST pull-down assay. Their co-location was observed under a laser scanning cofocal microscope. Histone H3K9 methyltransferase kit was used to check the methyltransferase activity, and Western blotting was used to detect the tri-methylation of histone H3K9. The effects of MTlh on proliferation and migration of cancer cells were detected by flow cytometry, colony formation, wound healing and borden chamber assays, respectively. Results： MTlh was directly bound to EHMT1, and they co-localized in the nuclei. MTlh expression increased the activity of histone methyltransferase, and it was associated wih histone H3K9 tri-methylation. Lung cancer cells with MTlh expression had higher percentage of Go cells, and lower movement and migration abilities. Conclusion; MTlh directly binds with EHMT1 in nuclei of lung cancer cells, and maybe regulate the methylation of histone H3K9 as a tumor supressor gene.
出处
《肿瘤》
CAS
CSCD
北大核心
2011年第1期4-10,共7页
Tumor
基金
国家自然科学基金资助项目(编号:300971114)
