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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 observe the interaction and localization of euchromatic histone methyltransferase 1 (EHMT1) and metallothionein 1h (MT1h), and to investigate its effect on histone H3K9 methylation and lung cancer cell biology The impact of behavior and possible mechanism of action. Methods: The pGBKT7-MT1h plasmid and pGADT7-EHMT1 plasmid were co-transformed into yeast strain AH109 for yeast two-hybrid assay. The lung cancer cell lines A549 and A2 were co-transfected with pcDNA4 / TO / myc-hisA / MT1h and pcDNA6 / TR plasmids and MT1h was induced by tetracycline. MT1h, c-myc and EHMT1 protein expression and localization; using histone H3K9 methyltransferase kit detection of methylation transferase activity, Western blot detection of trimethylated histone H3K9 expression; FCM and clone formation assay MT1h expression on cell proliferation The scratch test and Borden chamber method were used to detect the effect of MT1h on cell migration. Results: The experiment confirmed that MT1h and EHMT1 have a direct effect, and the two are located in the nucleus. After induction of MT1h expression, histone methyltransferase activity increased, and MT1h and histone lysine H3K9 trimethylation are closely related. Functional analysis showed that MT1h stably transfected lung cancer cells in the G0 phase, decreased cell motility and migration. CONCLUSION: MT1h and EHMT1 play a direct role in the nucleus and may play a role in tumor suppressor gene regulation by regulating histone H3K9 methylation.