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[摘要]
目的:研究抑制促分裂原活化蛋白激酶/细胞外信号调节激酶激酶3(mitogenactivated protein/extracellular signal regulated kinasekinase 3,MEKK 3)基因表达促进TRAIL诱导乳腺癌MCF7细胞凋亡的作用,寻找乳腺癌临床治疗新策略。方法:应用MTT法检测人TRAIL对MCF7细胞生长的抑制作用。合成MEKK3siRNA,应用脂质体介导MEKK3siRNA转染入人乳腺癌细胞MCF7,以RTPCR和Western blotting法检测MCF7细胞MEKK 3 mRNA和蛋白的表达。应用MTT法和流式细胞术检测MEKK3siRNA与TRAIL联合处理后MCF7细胞的增殖和凋亡。结果:TRAIL具有抑制MCF7细胞增殖作用,但其抑制作用较弱。MEKK3siRNA转染后能有效而稳定地抑制MCF7细胞中 MEKK3 mRNA和蛋白的表达(P<0.01)。TRAIL与MEKK3siRNA联合处理MCF7细胞较TRAIL单独处理更明显地抑制细胞增殖活力(P<0.05),更明显地增加细胞凋亡率(P<001)。结论:siRNA沉默 MEKK3基因能显著促进TRAIL对乳腺癌MCF7细胞凋亡的诱导作用,为探讨乳腺癌治疗新方案提供了实验依据。
[Key word]
[Abstract]
Objective:To investigate the promoting effect of silencing mitogenactivated protein/ERK kinase kinase 3 ( MEKK3 )gene on TRAIL-induced apoptosis of breast cancer MCF7 cells, so as to search for a novel clinical treatment strategy for breast cancer. Methods: TRAIL was used to treat MCF7 cells and the growth inhibition of MCF7 cells was determined by a methyl thiazolyl tetrazolium ( MTT ) assay.Chemically synthesized small interfering RNA ( siRNA ) targeting MEKK3 was transfected into MCF7 cells using DharmaFECT Transfection reagent, and the expression of MEKK3mRNA and protein was detected by RTPCR and Western blotting. The proliferation and apoptosis of MCF7 cells were analyzed by MTT and flow cytometry ( FCM ) after treatment with MEKK3siRNA combined with TRAIL.Results: TRAIL inhibited the proliferation of MCF7 cells, but the inhibitory effect was weak. Transfection with MEKK3siRNA effectively and stably inhibited the expression of MEKK3mRNA and protein expression (P<0.01). Combination of TRAIL and MEKK3siRNA more severely inhibited the proliferation of MCF7 cells compared with TRAIL alone (P<0.05); besides, the combination also increased the apoptosis rate of MCF7 cells (P<0.05). Conclusion: Silencing of MEKK3gene with siRNA can greatly promote TRAILinduced apoptosis of breast cancer cells MCF7, which lays an experimental foundation for new treatment method of breast cancer.
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[基金项目]
上海交通大学科技发展基金资助项目(No.05xj21022)。