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ECT2基因沉默调控人乳腺癌细胞增殖和凋亡及其机制探讨
         
ECT2 gene-silencing regulates the proliferation and apoptosis of human breast cancer cells and its mechanism

摘    要
目的:探讨上皮细胞转化序列2癌基因(epithelial cell transforming sequence 2 oncogene,ECT2)沉默对乳腺癌细胞增殖和凋亡的影响,以及其潜在的作用机制。
方法:首先采用实时荧光定量PCR和蛋白质印迹法检测正常乳腺上皮细胞(MCF-10A)和乳腺癌细胞(MDA-MB-231、SK-BR-3、MCF-7和BT474)中ECT2 mRNA及蛋白的表达水平。将特异性靶向沉默ECT2基因的siRNA(ECT2 siRNA)转染至乳腺癌MDA-MB-231和MCF-7细胞,并用细胞外调节蛋白激酶(extracellular regulated protein kinase,ERK)通路激活剂处理或者微RNA-101(microRNA-101,miR-101)抑制子转染后,采用CCK-8和FCM法分别检测细胞增殖和凋亡情况,采用实时荧光定量PCR和蛋白质印迹法检测细胞中磷酸化ERK (phospho-ERK,p-ERK)、Ras相关的C3肉毒底物1(Ras-related C3 botulinum toxin substrate 1,Rac1)和miR-101的表达水平。
结果:与正常乳腺上皮细胞相比,乳腺癌细胞中ECT2 mRNA和蛋白的表达水平均明显升高(P值均<0.05)。ECT2基因沉默后,乳腺癌细胞增殖被明显抑制(P<0.05),细胞凋亡被明显促进(P<0.05),并且细胞中p-ERK和Rac1表达被明显下调(P值均<0.05),而miR-101表达被明显上调(P<0.05)。ERK激活剂作用后,ECT2 siRNA转染对p-ERK、miR-101和Rac1表达的影响被明显反转(P值均<0.05);miR-101抑制子转染后,ECT2 siRNA转染对细胞增殖、凋亡以及Rac1表达的影响被反转(P值均<0.05),而对p-ERK表达没有明显影响(P>0.05)。
结论:ECT2基因沉默可能通过ERK/miR-101/Rac1信号转导通路,调节乳腺癌细胞的增殖和凋亡。
标    签 乳腺肿瘤   基因沉默   细胞增殖   细胞凋亡   上皮细胞转化序列2癌基因   细胞外调节蛋白激酶   微RNA-101   Breast neoplasms   Gene silencing   Cell proliferation   Apoptosis   Epithelial cell transforming sequence 2 oncogene   Extracellular regulated protein kinase   MicroRNA-101  
 
Abstract
Objective: To investigate the effects of epithelial cell transforming sequence 2 oncogene (ECT2) gene-silencing on the proliferation and apoptosis of human breast cancer cells, as well as its potential molecular mechanism.
Methods: Firstly, the expressions of ECT2 mRNA and protein in normal mammary epithelial cells (MCF-10A) and breast cancer cells (MDA-MB-231, SK-BR-3, MCF-7, and BT474) were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. After transfection with the specific siRNA targeting ECT2 gene (ECT2 siRNA) and treatment with extracellular regulated protein kinase (ERK) pathway activator or transfection with microRNA-101 (miR-101) inhibitor, the proliferation and apoptosis of MDA-MB-231 and MCF-7 cells were detected by CCK-8 and FCM assay, respectively. Then the levels of phospho-ERK (p-ERK), Ras-related C3 botulinum toxin substrate 1 (Rac1) and miR-101 in MDA-MB-231 and MCF-7 cells were detected by Western blotting and real-time fluorescent quantitative PCR.
Results: The expression levels of ECT2 mRNA and protein in breast cancer cells were significantly increased as compared with those in normal mammary epithelial cells (both P < 0.05). After ECT2 gene-silencing, the proliferation of MDA-MB-231 cells was inhibited (P < 0.05), the apoptosis was improved (P < 0.05), as well as the expressions of p-ERK and Rac1 were reduced (both P < 0.05), but the expression of miR-101 was increased (P < 0.05). After treatment with ERK activator, the effects of ECT2 gene-silencing on the levels of p-ERK, miR-101 and Rac1 were reserved (all P < 0.05); whereas, miR-101 inhibitor reserved the effects of ECT2 gene-silencing on the proliferation and apoptosis, as well as the levels of miR-101 and Rac1 in MDA-MB-231 cells (all P < 0.05), but the effect of ECT2 gene-silencing on p-ERK expression was not changed (P > 0.05).
Conclusion: ECT2 gene-silencing may affect the proliferation and apoptosis of breast cancer cells by ERK-miR-101-Rac1 signaling pathway.

中图分类号 R737.9   DOI 10.3781/j.issn.1000-7431.2017.11.119

 
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收稿日期 2017/3/10

修改稿日期 2017/4/12

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引用该论文: XIAO An,PENG Rongrong. ECT2 gene-silencing regulates the proliferation and apoptosis of human breast cancer cells and its mechanism[J]. Tumor, 2017, 37(6): 594~603
肖安,彭蓉蓉. ECT2基因沉默调控人乳腺癌细胞增殖和凋亡及其机制探讨[J]. 肿瘤, 2017, 37(6): 594~603


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