转录因子GRHL3抑制SNX16表达促进乳腺癌细胞迁移和侵袭

GRHL3 promotes breast cancer cells migration and invasion via downregulation of SNX16

目的研究转录因子GRHL3参与调控分拣连接蛋白16(SNX16)表达的分子机制及其对乳腺癌细胞迁移和侵袭能力的影响。方法免疫组织化学检测乳腺癌组织和癌旁组织中GRHL3和SNX16的表达变化;通过建立过量表达GRHL3的乳腺癌细胞MCF7,Western blot检测GRHL3、SNX16在MCF7细胞中表达变化;利用分子克隆技术构建含有SNX16基因启动子和突变的SNX16基因启动子的荧光素酶报告基因表达载体,通过检测荧光素酶活性变化观察GRHL3对SNX16基因启动子的调控作用;利用Transwell迁移和侵袭实验检测GRHL3过量表达的MCF7细胞迁移和侵袭能力的影响,并通过转染SNX16 cDNA的表达载体回补SNX16表达,观察MCF7细胞的迁移和侵袭能力变化。结果免疫组织化学结果显示:与癌旁组织相比,乳腺癌组织中GRHL3高水平表达,而SNX16的表达水平较低;Western blot结果显示,过量表达GRHL3的MCF7,SNX16的表达水平显著降低(P<0.05);通过分子克隆技术成功构建SNX16基因启动子和突变的SNX16基因启动子的荧光素酶报告基因表达载体;荧光素酶活性检测实验结果显示GRHL3对荧光素酶报告基因SNX16启动子具有显著抑制作用,突变后GRHL3结合位点,GRHL3对SNX16基因启动子的负调控作用消失;Transwell迁移和侵袭实验结果说明过表达GRHL3的MCF7细胞侵袭和迁移能力显著增强,回补SNX16后,MCF7细胞侵袭和迁移能力明显减弱。结论在乳腺癌细胞中,转录因子GRHL3对SNX16的启动子具有负调控作用,GRHL3通过结合SNX16基因启动子抑制SNX16表达,促进细胞的迁移和侵袭。

Objective To investigate GRHL3 controlling SNX16 expression in breast cancer cells,and promoting cell migration and invasion.Methods Immuno-histochemistry analysis was performed to detect GRHL3 and SNX16 expression in breast cancer tissues.Western blot was used to determine GRHL3 and SNX16 expression in the MCF7 cells overexpressed GRHL3.Luciferase reporter vectors with the wild type or the mutated promoter of SNX16 gene were established using molecular cloning technology.Luciferase assay was used to detect activity of the SNX16 promoter in presence of or absence of GRHL3.Transwell analysis was performed to investigate cell migration and cell invasion.Results Immunohistochemistry analysis showed that GRHL3 overexpressed in breast cancer tissues,whereas SNX16 expressed at lower level in breast cancer tissues than that in precancerous lesions.Western blot analysis showed GRHL3 overexpression inhibited SNX16 expression in MCF7 cells.Sequencing results proved that the luciferase reporter vectors with the wild type or the mutated promoter of SNX16 gene were successfully established.Luciferase assay showed GRHL3 dramatically inhibited activity of the SNX16 promoter.Tanswell analysis indicated GRHL3 promoted MCF7 cells migration and invasion partly related to downregulation expression of SNX16.Conclusion GRHL3 promotes migration and invasion of breast cancer cells via downregulation of SNX16 expression.