RNA干扰YAP基因对人乳腺癌MDA-MB-231细胞生物学行为的影响

Effect of RNA interference targeting YAP gene onbreast cancer MDA-MB-231 cells

关键词:  乳腺肿瘤  Yes相关蛋白  siRNA  细胞功能

Key words:  breast cancer  Yes associated protein  siRNA  cellular function

摘要:

目的 探讨RNA干扰YAP基因对人乳腺癌MDA-MB-231细胞生物学行为的影响。方法 使用阳离子脂质体转染试剂LipofectamineTM2000将靶向沉默YAP基因的siRNA序列转染至乳腺癌MDA-MB-231细胞中,采用qRT-PCR和Western印迹法分别检测转染后MDA-MB-231细胞中YAP基因及蛋白的表达水平,噻唑蓝(MTT)实验和细胞平板克隆实验检测转染前后细胞增殖的变化,Transwell小室和划痕实验观察转染对细胞侵袭及迁移的影响,流式细胞术评价转染后细胞周期及凋亡的变化情况。结果 转染siRNA后,YAP RNA和蛋白的表达量相对于空白对照及阴性对照组均明显下降(P<0.01)。MTT实验及细胞平板克隆实验显示,siRNA干扰YAP表达可以显著抑制乳腺癌MDA-MB-231细胞的增殖活性；Transwell小室及划痕试验显示,siRNA干扰YAP的表达可以明显抑制乳腺癌MDA-MB-231细胞的侵袭及迁移能力；细胞周期实验显示,沉默YAP后,细胞周期出现G₀/G₁期阻滞,细胞凋亡检测证实沉默YAP后细胞凋亡率并未出现明显上升。结论 抑制YAP在乳腺癌细胞的表达可有效降低细胞的增殖、迁移和侵袭能力,改变细胞周期分布,但对细胞凋亡无明显影响。

Abstract:

Objective To investigate the effect of small interfering RNA (siRNA) targeting YAP gene on breast cancer MDA-MB-231 cells. Methods siRNA targeting YAP gene was transfected in human breast cancer MDA-MB-231 cells with LipofectamineTM2000. The effect of siRNA on cell proliferation was assessed by MTT assay; cell migration and invasion were examined by colony formation assay, Transwell migration assay and wound healing assay; cell cycle and apoptosis were evaluated by flow cytometry; the expression of YAP mRNA and protein in MDA-MB-231 cells was detected by quantitative Real-Time-polymerase chain reaction (qRT-PCR) and Western blotting analysis, respectively. Results Expression of YAP mRNA and protein was suppressed after trans-fected with siRNA verified by qRT-PCR and Western blotting (P<0.01). MTT assay and colony formation assay showed that the proliferation of MDA-MB-231 cells was inhibited in transfected MDA-MB-231 cells compared with blank control and negative control. Transwell and wound healing test demonstrated that migration and invasion of MDA-MB-231 cells were significantly inhibited. Flow cytometry showed that MDA-MB-231 cells arrested at G₀/G₁ phase but cell apoptosis was not changed after iRNA interference. Conclusion This study demonstrates that YAP gene may play important role in biological behaviors of human breast cancer MDA-MB-231 cells.