miR-449a在乳腺癌裸鼠皮下移植瘤模型中的作用

摘要

Objective To investigate the effects of knockdown of miR-449a on breast cancer xenograft in nude mice.Methods In this study,we established a model of breast cancer xenograft in nude mice with breast cancer MCF-7 cell line,then stably transfected MCF-7 cells with plasmids containing shRNA-miR-449a precursors;the stable transfected cells were injected subcutaneously into the nude mice by multi-point injection.We used negative plasmid as negative control and PBS as blank control.We detected tumor volume after the treatment of nude mice and tumor growth inhibition rate was calculated.The expressions of miR-449a and Notch1 in xenograft in nude mice were detected by real-time PCR;the levels of Notch 1,β-catenin and E-cadherin in the xenograft tumor tissues were detected by Western blot; and the expression of E-cadherin in tumor tissues was detected by immunohistochemistry.Results In the process of xenograft tumor formation,no nude mice died.Knockdown of miR-449a could significantly inhibit the growth of tumor volume after tumor detection (P <0.05 ).miR-449a was significantly down-regulated in xenograft tumor tissues,and the expressions of Notch 1 and β-catenin were also down-regulated,while the level of E-cadherin was increased.The results of immunohistochemistry showed that the expression of Notch1 protein was decreased and that of E-cadherin protein was increased (P <0.05).Conclusion Knockdown of miR-449a inhibited the normal growth of breast cancer xenografts in nude mice by down-regulating the expressions of Notch 1 andβ-catenin and promoting the expression of E-cadherin.%目的 探究敲低miR-449a对裸鼠皮下乳腺癌细胞移植瘤生长的影响.方法 本实验通过采用乳腺癌MCF-7细胞系建立乳腺癌的裸鼠皮下移植瘤动物模型,将含有shRNA-miR-449a表达的质粒稳定转染MCF-7细胞,将筛选的稳转细胞采取多点注射的方法注入裸鼠皮下,采用阴性质粒为阴性对照,PBS作为空白对照;处理裸鼠后检测成瘤体积并计算肿瘤生长抑制率;Real-time PCR检测肿瘤组织miR-449a、Notch 1表达情况;Western blot检测移植瘤组织内的Notch 1、β-catenin和E-cadherin蛋白水平;免疫组织化学技术检测肿瘤组织内Notch1、E-cadherin表达水平.结果 移植瘤形成过程中裸鼠未出现死亡,肿瘤体积检测后发现miR-449a敲低后能够显著抑制肿瘤的生长(P<0.05);miR-449a在移植瘤组织内表达降低,同时Notch 1蛋白表达降低,β-catenin表达降低,而E-cadherin蛋白表达升高;免疫组化结果也显示Notch 1蛋白水平降低,E-cadherin蛋白的表达水平升高(P<0.05).结论 敲低miR-449a可能是通过下调Notch 1、β-catenin蛋白水平,促进E-cadherin蛋白表达,从而抑制乳腺癌裸鼠皮下移植瘤的生长.