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目的通过研究小干扰RNA(small interfering RNA,siRNA)沉默人乳腺癌MDA-MB-231细胞中YAP基因的表达,观察YAP基因沉默后对人乳腺癌MDA-MB-231细胞功能的影响。方法使用阳离子脂质体转染试剂LipofectamineTM2000将靶向沉默YAP基因的siRNA序列转染至乳腺癌M DA-M B-231细胞中,采用实时定量逆转录聚合酶链反应(quantitative real time-polymerase chain reaction,qRT-PCR)和蛋白质印迹法(Western blot)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用来检测转染前后细胞增殖的变化,Transwell小室和划痕实验用来观察转染对细胞侵袭及迁移的影响,流式细胞术用来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结果显示,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M DA-M B-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。
OBJECTIVE: To investigate the effect of YAP gene silencing on the function of human breast cancer cell line MDA-MB-231 by silencing the expression of YAP gene in human breast cancer MDA-MB-231 cells by small interfering RNA (siRNA). Methods siRNA targeting silencing of YAP gene was transfected into breast cancer M DA-M B-231 cells using LipofectamineTM2000 as a transfection reagent. Quantitative real time-polymerase chain reaction The expression of YAP gene and protein in MDA-MB-231 cells was detected by qRT-PCR, qRT-PCR and Western blot, respectively. MTT and MTT assay were used to detect the expression of YAP gene and protein. Transwell chamber and scratch assay were used to observe the effect of transfection on cell invasion and migration. Flow cytometry was used to evaluate the changes of cell cycle and apoptosis after transfection. Results After transfected with siRNA, the expression of YAP RNA and protein were significantly decreased compared with the blank control group and the negative control group (P <0.01). MTT assay and cell plate cloning experiments showed that siRNA interference YAP expression can significantly inhibit the proliferation of breast cancer MDA-MB-231 cells, Transwell chamber and scratch test showed that siRNA interference YAP expression can significantly inhibit breast cancer M DA- M B-231 cell invasion and migration ability, cell cycle experiments showed that after YAP silencing, cell cycle G0 / G1 phase arrest, apoptosis test confirmed that silencing YAP cell apoptosis rate did not appear significantly increased. Conclusion Inhibition of YAP expression in breast cancer cells can effectively reduce the cell proliferation, migration and invasion ability, change the cell cycle distribution, but had no significant effect on apoptosis.