Slug真核表达载体构建及功能鉴定

目的 在肿瘤发生和发展过程中,上皮细胞-间质转化(epithelial-mesenchymal transition,EMT)是上皮来源恶性肿瘤细胞获得侵袭、转移能力的重要生物学过程,并且与乳腺癌干细胞的生物学特性密切相关.本研究通过构建Slug的慢病毒真核表达载体,探讨Slug基因与EMT之间的关系及对乳腺癌干细胞生物学特性的影响.方法 以军事医学科学院(生物工程研究所)保存的乳腺cDNA文库为模板,通过PCR扩增出Slug的全长编码序列,将其克隆到经BamH Ⅰ和EcoR Ⅰ酶切的PCDH表达载体上,构建成PCDH-Slug真核表达载体,在人293T细胞中转入空载体和重组质粒,利用蛋白质印迹法检测PCDH载体介导的Slug的表达.后将包装质粒共转染293T细胞,包装病毒,感染ZR75-1细胞,经Puro(嘌呤霉素)筛选2周后,得到稳定表达Slug的ZR75-1细胞株,检测ZR75-1细胞对EMT的影响.并将稳定表达Slug的ZR75-1稳定细胞接种于的Matrigel基质胶,倒置显微镜观察ZR75-1稳定细胞株的成管特性.结果 从乳腺文库中获得Slug的全长编码序列,经BamH Ⅰ、EcoR Ⅰ双酶切及DNA测序证实得到PCDH-Slug表达载体,PCDH-Slug真核表达载体和PCDH分别转染293T细胞后,经过蛋白质印迹法检测Slug在293T细胞内有显影,对照组明显减弱,表明Slug-PCDH载体携带的Slug编码序列能够在293T细胞中成功;通过增加puro的含量进行筛选,得到稳定表达的混合克隆即能表达Slug的ZR75-1细胞株,经蛋白质印迹法检测PCDH-Slug 对E-cadherin的影响,以GAPDH为内参,结果比对照组的表达减弱,与预期相符,表明PCDH-Slug载体可以使细胞中E-cadherin的表达减弱;倒置显微镜下ZR75-1稳定细胞株成管明显,表明Slug可以通过调节EMT使乳腺癌细胞具有某些干性特征.结论 调节Slug基因表达可以影响EMT相关蛋白的表达,并以此影响乳腺癌干细胞的生物学特性.

Construction and functional identification of the Slug eukaryotic expression vector

OBJECTIVE The epithelial-mesenchymal transition (EMT) can endow cancer cells with the migratory and invasive capabilities associated with metastatic competence and play an important role in the formation of breast cancer stem cells.This study aimed to investigate the relationship between Slug gene and EMT and the effect of Slug on breast cancer stem cells by constructing an eukaryotic expression vector of human Slug gene.METHODS The coding sequences of full length Slug was amplified from breast library by PCR and cloned into the PCDH vector.Slug expression was detected by Western blot after the recombinant plasmids were transfected into 293T cells.PCDH-Slug was then packaged with accessory plasmids into lentivirus in 293T cells and selected for 2 weeks with puro before the mixed colonies stably expressing Slug were obtained.The tube characteristics were detected by fluorescence microscope.RESULTS The PCDH-Slug expression vector was successfully obtained,ZR75-1 cells stably expressing PCDH-Slug vector were constructed.Compared to the empty vector,the expression of E-cadherin was decreased and the expression of tube was increased.The results indicate that Slug gene could modulate EMT by E-cadherin and regulate breast cancer stem cells.CONCLUSION Slug gene can affect the expression of EMT-associated proteins and participate in the regulation of breast cancer stem cells.