摘要
目的探讨miR-7-5p和miR-152-3p协同抑制乳腺癌细胞上皮-间质转化(epithelial-mesenchymal transition,EMT)进程及紫杉醇耐药的分子机制。方法采用生物信息学软件预测miR-7-5p和miR-152-3p的靶基因,双荧光素酶报告基因检测两者与TCF4的靶向关系;Western blot法检测各组细胞中Wnt/β-catenin信号通路关键调控因子β-catenin及TCF4蛋白的表达;在转染miR-7-5p mimics和miR-152-3p mimics基础上给予Wnt/β-catenin通路激活剂LiCl处理后,Western blot法检测MCF-7/TAX细胞中β-catenin、TCF4和EMT相关蛋白(E-cadherin、vimentin)的表达,Transwell小室实验检测MCF-7/TAX细胞侵袭和迁移能力;MTT实验检测激活Wnt/β-catenin信号通路对MCF-7/TAX细胞紫杉醇耐药性的影响。结果TCF43′UTR区域存在能够与miR-7-5p及miR-152-3p互补的结合位点;转染miR-7-5p mimics和miR-152-3p mimics后可使TCF4野生型(TCF4-WT)报告基因载体的荧光素酶活性较NC组明显降低(P<0.05)。Western blot结果显示,与NC组相比,转染miR-7-5p mimics和miR-152-3p mimics后各组紫杉醇耐药MCF-7/TAX细胞中β-catenin和TCF4蛋白的表达水平明显降低(P<0.05),且两者共同转染后MCF-7/TAX细胞中β-catenin和TCF4蛋白的表达水平较miR-7-5p组或miR-152-3p组进一步降低(P<0.05)。Western blot结果显示,LiCl处理后MCF-7/TAX细胞中β-catenin、TCF4和vimentin蛋白表达水平明显升高,而E-cadherin蛋白表达水平明显降低(P<0.05)。Transwell小室结果显示,LiCl处理后MCF-7/TAX细胞侵袭和迁移能力明显增强(P<0.05)。MTT结果显示,不同浓度紫杉醇作用下,miR-7-5p+LiCl组细胞增殖活力较miR-7-5p组明显升高;同时miR-152-3p+LiCl组和miR-7-5p/152-3p+LiCl组细胞的增殖活力较NC组均明显升高(P<0.05)。结论TCF4是miR-7-5p和miR-152-3p的共同靶标。miR-7-5p和miR-152-3p可共同抑制MCF-7/TAX细胞中Wnt/β-catenin信号通路的活化。
Purpose To explore the molecular mechanism of miR-7-5p and miR-152-3p synergistically inhibiting epithelial-mesenchymal transition(EMT)process and paclitaxel resistance in breast cancer cells,which is related to targeted regulation of Wnt/β-catenin signaling pathway.Methods Targetscan was used to predict the target genes of miR-7-5p and miR-152-3p.The targeting relationship between miR-7-5p,miR-152-3p and TCF4 was detected by double luciferase reporter gene.The expression levels of the key regulatory factors of Wnt/β-catenin signaling pathway,β-catenin and TCF4 protein were detected by Western blot.The expression ofβ-catenin,TCF4,EMT-related protein E-cadherin and vimentin in MCF-7/TAX cells was detected by Western blot after treatment with LiCl,a Wnt/β-catenin signal pathway activator,on the basis of transfection of miR-7-5p mimics and miR-152-3p mimics.The invasion and migration ability of MCF-7/TAX cells were detected by Transwell chamber.The effect of activating Wnt/β-catenin signaling pathway on paclitaxel resistance in MCF-7/TAX cells was detected by MTT.Results There were binding sites complementary to miR-7-5p and miR-152-3p in the TCF43′UTR region.The luciferase activity of TCF4-WT reporter gene vector was significantly decreased after transfection with miR-7-5p mimics and miR-152-3p mimics compared with the control NC group(P<0.05).Western blot results showed that the expression levels ofβ-catenin and TCF4 proteins in MCF-7/TAX cells transfected with miR-7-5p mimics and miR-152-3p mimics were significantly lower than those in control NC group(P<0.05),and the expression levels ofβ-catenin and TCF4 proteins in MCF-7/TAX cells co-transfected with miR-7-5p group or miR-152-3p group were further decreased(P<0.05).Western blot results showed that the expression levels ofβ-catenin,TCF4 and vimentin in MCF-7/TAX cells treated with LiCl increased significantly,while the expression levels of E-cadherin protein decreased significantly(P<0.05).Transwell results showed that the invasion and migration ability of MCF-7/TAX cells treated with LiCl was significantly enhanced(P<0.05).MTT results showed that the proliferation activity of miR-7-5p+LiCl group was significantly higher than that of miR-7-5p group at different concentrations of paclitaxel,and the proliferation activity of miR-152-3p+LiCl group and miR-7-5p/152-3p+LiCl group was also significantly higher than that of corresponding control NC group(P<0.05).Conclusion TCF4 is the common target of miR-7-5p and miR-152-3p.Both miR-7-5p and miR-152-3p can co-suppress the activation of Wnt/β-catenin signaling pathway in MCF-7/TAX cells.
作者
李娜
张哲莹
朱会芳
贺国洋
韩正华
原志庆
王凡平
王明永
LI Na;ZHANG Zhe-ying;ZHU Hui-fang;HE Guo-yang;HAN Zheng-hua;YUAN Zhi-qing;WANG Fan-ping;WANG Ming-yong(Department of Pathology,School of Laboratory Medicine,Xinxiang Medical University,Xinxiang 453003,China;College of Stomatology and Technology,Sanquan College of Xinxiang Medical University,Xinxiang 453003,China;Xinxiang Key Laboratory of Immunoregulation and Molecular Diagnostics,School of Laboratory Medicine,Xinxiang Medical University,Xinxiang 453003,China)
出处
《临床与实验病理学杂志》
CAS
CSCD
北大核心
2021年第7期765-770,共6页
Chinese Journal of Clinical and Experimental Pathology
基金
国家自然科学基金(31800658)
河南省教育厅高等学校重点科研项目(21A310014)
河南省高校科技创新团队支持计划项目(20IRTSTHN030)
新乡市科技攻关资助项目(GG2020039)。
