摘要
目的 探讨转化生长因子-β1(TGF-β1)通过诱导乳腺癌细胞上皮-间充质转化(EMT)影响细胞侵袭迁移能力及相关分子机制.方法 5μg/L TGF-β1作用于人乳腺癌细胞株MDA-MB-435S,诱导细胞发生EMT后,应用脂质体2000瞬时转染小干扰RNA(siRNA)沉默Smad2的表达,观察乳腺癌细胞的形态学变化,并通过Western blot检测Smad2静默前后细胞角蛋白、E-钙黏素、α-平滑肌肌动蛋白(α-SMA)、波形蛋白、Smad2、磷酸化Smad2(p-Smad2)的表达变化;通过Transwell实验检测细胞侵袭迁移能力的改变.结果 TGF-β1作用72h后,乳腺癌细胞株MDA-MB-435S部分细胞由多角形转变为成纤维样细胞形态,间充质细胞标志物α-SMA、波形蛋白表达分别上调了3.86倍和1.17倍(P<0.01);上皮细胞标志物细胞角蛋白、E-钙黏素蛋白表达分别下降了63%和70% (P <0.01),p-Smad2表达明显上调1.2倍(P<0.01);侵袭的细胞数(79.20 ±7.35)明显多于空白组(48.93±5.43),差异有统计学意义(P<0.05).沉默Smad2的表达后细胞角蛋白表达升高了44%(P<0.01),波形蛋白表达下降了59% (P <0.01),干扰组细胞侵袭数目(54.34 ±6.09)显著低于对照组(75.09 ±4.98),差异有统计学意义(P<0.05).结论 体外TGF-β1能够诱导乳腺癌细胞EMT,增强乳腺癌细胞侵袭转移能力;Smad2信号转导通路在乳腺癌细胞EMT中起重要作用.
Objective To investigate the effect of transforming growth factor-β1 (TGF-β1) on the invasion and metastasis ability of breast cancer cells through inducing epithelial-mesenchymal transition (EMT),and explore its related molecular mechanism.Methods The breast cancer cells MDA-MB-435S were treated with recombinant human TGF-β1 (5 μg/L).Then the cells were transfected with small inference RNA (siRNA) against Smad2 or control siRNA using Lipfectamine 2000.Western blotting was used to detect the expression of some epithelial markers,mesenchymal markers,Smad2 and phosphorylated Smad2 (p-Smad2).The Matrigel-coated Transwell was used to detect the invasion and metastasis ability of breast cancer cells.Results After TGF-β1 treatment for 72 h,MDA-MB-435S cells showed more elongated shape,displaying firoblast-like appearance compared to the untreated cells.Compared with the control group,the expression of α-smooth muscle actin and vimentin were increased by 3.86 and 1.17 times respectively (P 〈 0.01),the expression of cytokeratin and E-adherin were decreased by 63% and 70% respectively (P 〈0.01) and the expression of p-Smad2 was increased by 1.2 times compared with before (P 〈 0.01).The number of invasion cells in TGF-β1 group (79.20 ± 7.35) were markerly increased than non-treated group (48.93 ± 5.43,P 〈 0.05).After knockdown of the Smad2 gene partially,the expression of cytokeratin was increased by 44% (P 〈 0.01),the expression of vimentin was decreased by 59% (P 〈 0.01).The number of invasion cells in siRNA group (54.34 ± 6.09) was decreased as compared with control group (75.09 ± 4.98) (P 〈 0.05).Conclusion Our current data demonstrated that EMT of breast cancer cells induced by TGF-β1 promotes the migration and invasion of breast cancer cells.Smad2 signal transduction pathway may play an important role in the EMT process of breast cancer cells.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2015年第5期979-982,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目(81302290)