FOXP3通过下调NCKAP1的表达抑制乳腺癌转移

FOXP3 inhibits breast cancer metastasis by down-regulating NCKAP1

目的探讨FOXP3对乳腺癌转移的影响及分子机制。方法在乳腺癌细胞系MCF7中转染FOXP3质粒,进行高通量转录组测序以及基因差异表达分析。在T47D和MCF7中均过表达和敲低FOXP3,RT-qPCR检测NCKAP1的表达。利用Jaspar数据库分析NCKAP1基因的启动子,ChIP实验检测FOXP3与NCKAP1基因启动子的结合活性,报告基因实验检测不同FOXP3的表达水平对NCKAP1启动子转录活性的影响。单独si-FOXP3、si-FOXP3和si-NCKAP1、control siRNA分别转染T47D细胞,划痕实验和Transwell实验检测细胞的迁移和侵袭能力。在GEPIA数据库中分析乳腺癌病人NCKAP1的表达水平以及生存期。在TCGA和GEPIA数据库中分析乳腺癌病人FOXP3和NCKAP1基因的表达相关性。结果转录组测序结果表明,高表达FOXP3的MCF7细胞中NCKAP1表达下调。MCF7细胞和T47D细胞过表达FOXP3后,NCKAP1表达水平降低(P<0.05)。沉默FOXP3后,MCF7细胞(P<0.01)和T47D细胞(P<0.001)内NCKAP1表达水平升高。NCKAP1基因的启动子上存在FOXP3的潜在结合位点,T47D细胞内FOXP3与NCKAP1基因的启动子结合(P<0.01)。FOXP3抑制NCKAP1启动子的转录活性(P<0.001),且FOXP3表达水平越高抑制效果越明显(P<0.05)。沉默FOXP3后,T47D细胞的迁移(P<0.01)和侵袭(P<0.001)能力增强,同时沉默FOXP3和NCKAP1后,T47D细胞的迁移和侵袭能力降低(P<0.001)。NCKAP1高表达的乳腺癌患者预后较差(P<0.05)。FOXP3和NCKAP1的表达量呈负相关(r=-0.1563,P<0.001)。结论FOXP3通过与NCKAP的启动子结合下调其表达,从而抑制乳腺癌的转移。

Objective To investigate the effect of FOXP3 on the invasion and migration of human breast cancer cells and explore its mechanism.Methods High-throughput transcriptome sequencing and differential gene expression analysis were performed on MCF7 cells overexpressing FOXP3.After FOXP3 was overexpressed and knocked down in breast cancer cell lines T47D and MCF7 respectively,the expression level of NCKAP1 mRNA was analyzed by real time PCR.In the Jaspar database,the promoter region of NCKAP1 gene was analyzed,ChIP experiment was used to verify whether FOXP3 binds to the promoter of NCKAP1 gene,and the reporter gene experiment was used to detect the effect of different expression level of FOXP3 on the transcriptional activity of NCKAP1 promoter.After T47D cells were transfected with si-FOXP3,si-FOXP3 and si-NCKAP1,or negative control siRNA,respectively,the cell migration and invasion abilities were detected by cell scratch and Transwell assay.In GEPIA database,the expression level of NCKAP1 and survival time in breast cancer patients were collected.The correlation of FOXP3 and NCKAP1 gene expression in breast cancer patients was analyzed in TCGA and GEPIA database.Results Transcriptome sequencing showed that FOXP3 down-regulated the expression of NCKAP1.After overexpressing FOXP3 in MCF7 and T47D cells,the expression levels of NCKAP1 were significantly decreased(P<0.05).After silencing FOXP3,the expression levels of NCKAP1 were significantly increased in MCF7(P<0.01)and T47D cells(P<0.001).There were potential FOXP3 binding sites on the promoter of NCKAP1 gene.In T47D cells,FOXP3 bound to the promoter of NCKAP1 gene(P<0.01).FOXP3 inhibited the transcriptional activity of NCKAP1 promoter(P<0.001),and the inhibition was stronger when the FOXP3 level was higher(P<0.05).After silencing FOXP3,the migration(P<0.01)and invasion(P<0.001)abilities of T47D were significantly enhanced.After both FOXP3 and NCKAP1 were silenced,the migration and invasion abilities of T47D were significantly reduced(P<0.001).Breast cancer patients with high NCKAP1 level had a poor prognosis(P<0.05).FOXP3 and NCKAP1 expression levels were negatively correlated in breast cancer patients(r=-0.1563,P<0.001).Conclusion FOXP3 may down-regulate the expression of NCKAP1 through binding to NCKAP1 promoter,thus inhibiting the metastasis of breast cancer.