二甲双胍可能通过Hippo-YAP通路抑制HER-2阳性乳腺癌细胞的增殖和促进凋亡

目的 观察二甲双胍对HER-2阳性乳腺癌细胞株SKBR3增殖、凋亡的影响，并探讨其可能的机制。方法 分别用0、20、40、60、80、100、120 μmol/L二甲双胍处理乳腺癌细胞株SKBR3，CCK-8法检测其对细胞增殖的影响；结晶紫染色观察其对细胞集落形成的影响；计算IC50值。以该浓度二甲双胍处理细胞，与空白对照相比，流式细胞术检测细胞凋亡和周期的改变；实时荧光定量 PCR（qRT-PCR）检测 YAP、TAZ、EGFR、CTGF、CYR61、E-cadherin、N-cadherin、Vimentin 及 Fibronectin 等关键基因的mRNA水平表达；Western blotting 实验检测YAP、TAZ蛋白水平的表达；免疫荧光观察二甲双胍对SKBR3细胞中YAP/TAZ核易位的改变。结果 CCK-8和细胞克隆实验显示二甲双胍对SKBR3细胞增殖活力具有明显的抑制作用（P<0.05），呈浓度和时间依赖性。与对照组相比，二甲双胍处理后的SKBR3细胞凋亡明显增加；G1期细胞比例增多，而G2/M期细胞比例减少。N-cadherin、Vimentin和Fibronectin的表达降低，而E-cadherin的表达上调（P<0.05）。qRT-PCR和Western blotting检测结果显示，二甲双胍处理后YAP、TAZ、EGFR、CTGF和CYR61的表达明显下调（P<0.05）。免疫荧光实验显示：实验组较对照组的YAP或TAZ，其荧光的定位更多地聚集于胞浆，而细胞核中荧光的量明显的减少。结论 二甲双胍能抑制HER-2阳性乳腺癌细胞SKBR3的增殖，促进其凋亡和上皮－间质转化，其机制可能与抑制YAP/TAZ的表达和核定位有关。

Metformin inhibits proliferation and promotes apoptosis of HER-2 positive breast cancer cells possibly through the Hippo-YAP pathway

Objective To investigate the effect of metformin on the proliferation and apoptosis of HER-2-positive breast cancer cell line SKBR3 and explore the possible mechanism of its action. Methods SKBR3 cells were treated with different concentrations (20-120 μmol/L) of metformin, and the changes in cell proliferation and colony formation ability were assessed using CCK-8 assay and crystal violet staining, respectively. Flow cytometry was performed to analyze cell apoptosis and cell cycle changes. Real-time fluorescent quantitative PCR (qRT-PCR) was used to detect mRNA expressions of YAP, TAZ, EGFR, CTGF, CYR61, E-cadherin, N-cadherin, vimentin and fibronectin in the treated cells, and the protein expressions of YAP and TAZ were detected using Western blotting; immunofluorescence assay was used to observe YAP/TAZ nuclear translocation in the cells. Results Metformin treatment significantly inhibited the proliferation of SKBR3 cells (P<0.05) in a concentration- and time-dependent manner. The results of flow cytometry showed that metformin significantly promoted apoptosis and caused cell cycle arrest at G1 phase in SKBR3 cells. Metformin treatment significantly down-regulated the mRNA expressions of YAP, TAZ, EGFR, CTGF and CYR61, N-cadherin, vimentin and fibronectin (P<0.05) and up-regulated the expression of E-cadherin (P<0.05); Western blotting results showed that YAP and TAZ protein expressions were significantly down-regulated in the cells after metformin treatment (P<0.05). Immunofluorescence assay revealed that metformin treatment caused the concentration of YAP and TAZ in the cytoplasm, and significantly reduced their amount in the cell nucleus. Conclusion Metformin can inhibit proliferation and promote apoptosis and epithelal-mesenchymal transition of HER-2 positive breast cancer cells possibly by that inhibing YAP and TAZ expression and their nuclear localization.