人乳腺癌细胞系MCF-7中FOXM1调控SYK转录

FOXM1 regulates transcription of SYK in breast cancer cell line MCF-7

目的探讨人乳腺癌细胞系MCF-7中叉头框转录因子M1(FOXM1)对脾酪氨酸激酶(SYK)表达的调控机制。方法从MCF-7、T47D、SK-BR3和MDA-MB-231中提取RNA和蛋白质,分别用PCR和Western blotting检测SYK的表达水平,并检测SYK基因启动子区CpG岛甲基化。在MCF-7中过表达FOXM1,采用Western blotting和实时荧光定量PCR检测FOXM1和SYK的表达。利用染色质免疫沉淀(ChIP)技术和荧光素酶报告基因检测转录因子FOXM1对SYK基因转录水平的调控作用。结果 SYK在MCF-7中呈阳性表达,而在T47D、SKBR3和MDA-MB-231中呈阴性表达;MCF-7细胞中SYK基因启动子区Cp G岛甲基化水平低于对照细胞(P<0.05)。在MCF-7中过表达FOXM1能下调SYK表达,在SK-BR3细胞系中沉默FOXM1基因能激活SYK表达。ChIP结果显示FOXM1直接参与调控SYK的转录;基因沉默FOXM1激活SYK基因启动子活性,过表达FOXM1抑制SYK基因启动子的活性。结论在人乳腺癌细胞系MCF-7中,SYK是FOXM1的靶基因,FOXM1可能通过调节SYK的表达来调控乳腺癌的发生发展。

Objective To explore the underlying mechanism of Forkhead box M1（FOXM1） regulating spleen tyrosine kinase（SYK） expression in breast cancer cell line MCF-7.Methods PCR and Western blotting assay were used to analyze the mRNA and protein levels of SYK in MCF-7,T47 D,SKBR3 and MDA-MB-231 cells.The methylation status of SYK promoter CpG island was detected.FOXM1 was overexpressed in MCF-7 cells,and then mRNA and protein levels of SYK and FOXM1 were detected.Chromatin immunoprecipitation（ChIP） assay and luciferase reporter assay were adopted to examine whether FOXM1 directly regulated the promoter activity of SYK.Results MCF-7 cells expressed endogenous SYK while T47 D,SK-BR3 and MDA-MB-231 cells did not.The rate of DNA methylation of SYK in MCF-7 cells was lower than that in control cells（P〈0.05）.Overexpression of FOXM1 decreased SYK expression in MCF-7 cells and silence of FOXM1 in SK-BR3 cells increased SYK expression.ChIP assay indicated that FOXM1 directly regulated the transcriptional level of SYK gene.Knockdown of FOXM1 increased the basal SYK promoter activity and overexpression of FOXM1 decreased luciferase activity of SYK in MCF-7 cells.Conclusion SYK is a target gene of FOXM1 in MCF-7,while FOXM1 is involved in the genesis and development of breast cancer by directly regulating SYK.