摘要
目的观察S100A6基因沉默对人食管癌Eca109细胞增殖活性及迁移能力的影响。方法查找人S100A6mRNA序列,设计针对其编码区的siRNA序列;根据siRNA序列设计合成shRNA并构建shRNA重组表达载体;用阳离子脂质体转染重组质粒至Eca109细胞,于转染后48h,采用real-time PCR法检测细胞中S100A6mRNA表达量,蛋白质印迹法检测细胞中S100A6蛋白表达量;MTT法检测细胞增殖活性,绘制细胞生长曲线;通过细胞划线法检测基因沉默对于肿瘤细胞迁移能力的影响。结果成功构建了针对S100A6基因的shRNA真核表达载体;通过脂质体转染的方法可在Eca109细胞内高效沉默S100A6,细胞转染后48h,与未转染细胞比较,S100A6 mRNA表达量降低,且差异有统计学意义(P<0.05,P<0.01),蛋白表达量检测结果与mRNA检测结果一致;S100A6基因沉默可明显抑制对数生长期Eca109细胞增殖活性,与未转染细胞比较,差异有统计学意义(P<0.01);S100A6基因沉默可明显抑制对数生长期Eca109细胞迁移能力。结论通过shRNA表达载体途径可在Eca109细胞中有效沉默S100A6基因,S100A6基因沉默能够显著抑制Eca109细胞增殖活性及迁移能力。
Objective To investigate the effects of S100A6 gene silencing on the proliferation and migration of Ecal09 human esophagus cancer ceils. Methods The shRNA expression vectors were constructed using the shRNA sequences designed based on human S100A6＇s coding sequence, and were transfected into Ecal09 cells via cationic liposome. The changes of S100A6 mRNA and protein in Ecal09 cells transfected with the recombinant vectors were detected using real-time PCR and Western blotting analysis 48 hours after transfection, respectively; the proliferative curves of transfected cells were plotted using MTT assay; furthermore, the change in cellular migration ability was determined using wound healing assay. Results The eukaryotic expression vector of shRNA targeting S100A6 was successfully constructed. Real-time PCR and Western blotting analysis results showed that S100A6 was effectively silenced by liposome-mediated transfection of the recombinant shRNA vectors in Ecal09 cells. Compared with the untransfected cells, S100A6 mRNA and protein in transfected Ecal09 cells were significantly decreased （P〈0. 05, P〈0. 01）. Meanwhile, the proliferative activity of Ecal09 cells was significantly inhibited by S100A6 silencing （P〈0.01）. It was found that the cellular migration was also suppressed by S100A6 gene interference. Conclusion S100A6 gene can be effectively silenced by shRNA expression vectors, and the silence may lead to inhibition of the proliferation and migration of Ecal09 cells.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2014年第1期49-54,共6页
Academic Journal of Second Military Medical University
