TGF-β1诱导乳腺癌细胞上皮-间充质转化中相关miRNAs筛选

摘要

Objective:To obtain the optimal concentration of TGF-β1 and screen for miRNAs that potentially involved in TGF-β1-induced epithelial-mesenchymal transition (EMT) in MCF-7 breast cells.Methods:Epithelial-mesenchymal transition (EMT) in MCF-7 breast cells was induced by different concentrations of TGF-31 for 48h.The morphology of MCF-7 cells under TGF-β1 treatment was observed by optical microscope.The EMT-related genes in MCF-7 cells were further determined by Real-time PCR and Western blot,and then several EMT-related miRNAs were selected to be verified by Real-time PCR.Results:The morphology in MCF-7 cells treated with TGF-β1 for 48h was shift from cobble to spindle-like shape.The results of Real-time PCR showed that the expression of Vimentin,par6,Col4A1,o-SMA and FN were upregulated,while the E-cadherin was downregulated after treated with TGF-β1 at 2,5 and 10ng/ml.Western blot results showed that the E-cadherin expression was downregulated,while the Vimentin and α-SMA expression were upregulated.Meanwhile,these results showed that the TGF-β1 at 10ng/ml exhibited a more significant effect than at 2 and 5ng/ml.In addition,the miR-16,miR-196a,miR-196b and miR-21 expressions were significantly upregulated after 10ng/ml TGF-β1 treatment for 48h.Conclusion:The EMT model of MCF-7 cell is successfully induced by TGF-β1 at 10ng/ml for 48h.The miR -16,miR-196a,miR-196b and miR-21 expressions are upregulated in TGF-β1-induced EMT cell model,which demonstrated that these miRNAs are closely associated with TGF-β1-induced EMT.%目的:探讨转化生长因子β1(TGF-β1)诱导乳腺癌细胞MCF-7上皮-间充质转化(epithelial-mesenchymal transition,EMT)的最佳浓度以及miRNAs在其中的差异表达.方法:不同浓度的TGF-β1作用于MCF-7细胞48h,光学显微镜下观察MCF-7细胞形态学的变化,同时Real-time PCR检测MCF-7细胞中上皮和间充质标志物表达变化;进一步利用Real-time PCR对通过生物信息学挑选的差异表达的miRNAs进行验证.结果:10ng/ml的TGF-β1作用MCF-7细胞48h后,能诱导MCF-7细胞发生EMT转化,同时筛选出与EMT密切相关的4个miRNAs(miR-16,miR-196a,miR-196b和miR-21).结论:miR-16、miR-196a、miR-196b和miR-21在TGF-β1诱导的EMT细胞模型中表达上调,发现这4个miRNAs与TGF-β1诱导的EMT密切相关.