MEK基因在黑米花青素抗HER-2阳性乳腺癌转移中的作用研究

The Role of Mitogen-Activated Protein Kinase(MEK) in the Anti-Metastasis Effect of Black Rice Anthocyanins Against HER-2 Positive Breast Cancer

目的探究丝裂原活化蛋白激酶激酶(mitogen-activated protein kinase kinase,MEK)基因在黑米花青素(black rice anthocyanins,BRACs)抗HER-2阳性乳腺癌转移中的作用。方法MDA-MB-453细胞经过BRACs、MEK抑制剂U0126、SiRNA沉默MEK单独或联合处理24h,采用划痕愈合实验、Transwell方法检测细胞迁移及侵袭能力,使用免疫沉淀法、免疫印迹法检测MEK磷酸化水平及其与HER-2蛋白的相互作用,利用RTPCR检测MEK mRNA在MDA-MB-453细胞中的表达。结果与对照组比较,BRACs、U0126及MEK-SiRNA单独或联合处理的实验组均能有效抑制MDA-MB-453细胞转移及侵袭能力,差异有统计学意义(P<0.05)。MEK蛋白磷酸化水平经BRACs处理后显著降低,MEK蛋白与HER-2蛋白之间的相互作用减弱,U0126联合BRACs处理MDA-MB-453细胞较BRACs单独处理更明显地抑制MEK磷酸化水平(P<0.05)。MDA-MB-453细胞MEK mRNA和蛋白的表达水平经MEK-SiRNA、BRACs单独或联合处理后均下调,差异有统计学意义(P<0.05)。结论 MEK基因与HER-2阳性乳腺癌的侵袭、转移有密切关系,其在BRACs抗HER-2阳性乳腺癌转移中起重要作用。

Objective To explore the role of MEK （mitogen-activated protein kinase kinase）in the anti- invasion of black rice anthocyanins（BRACs）against HER-2 positive breast cancer MDA-MB-453 cells. Methods BRACs,MEK inhibitor U0126 or MEK gene silencing were used individually or together to treat HER-2 positive breast cancer MDA-MB-453 cells for 24 h. Migration and invasion were detected by healing assay, cell migration assay and transwell method. Phosphorylation of MEK was analyzed by western blot. Interaction between HER-2 receptor and the protein of MEK was determined by immunoprecipitation. The expression of MEK gene mRNA was detected by RT-PCR. Results Compared with the control group, statistically significant decrease was observed in BRACs group, MEN inhibitor group, MEK-SiRNA group and co-treatment group on items of migration and invasion in MDA-MB-453 cells（P〈0.05） ;BRACs could significantly inhibit the phosphorylation level of MEK and weaken the interaction between HER-2 and the protein of MEK, while co-treatment group with both substances produced more remarkable inhibition effects （P〈0. 05）. BRACs group, MEK-SiRNA group and co-treatment group down regulated the mRNA expression of MEK and the protein of MEK（P〈0.05）. Conclusion MEK gene can greatly promote the migration/invasion of HER-2 positive breast cancer, which lays an experimental foundation for new treatment method for anti-metastasis using BRACs in HER-2 positive breast cancer.