靶向Y-box结合蛋白-1基因的shRNA质粒的构建及鉴定

Construction and Identification of shRNA Plasmid Targeting Y-Box Binding Protein-1 Gene

目的构建靶向Y-box结合蛋白-1(Y-box binding protein-1,YB-1)基因的短发夹RNA(shRNA)表达质粒并鉴定其抑制率。方法设计并合成3对针对YB-1基因的shRNA链,退火形成双链,与酶切的质粒pGenesil-1连接,构建3个重组质粒pGSi1、pGSi2和pGSi3,脂质体法转染人乳腺癌MDA-MB-231细胞,通过荧光定量PCR和Western blot分别检测细胞中YB-1基因mRNA的转录水平及蛋白的表达水平。结果重组质粒pGSi1、pGSi2和pGSi3经酶切及测序证明构建正确;转染MDA-MB-231细胞后,YB-1基因在mRNA和蛋白水平的表达均降低,其中pGSi2的抑制效果最好,在mRNA和蛋白水平的抑制率分别为57.4%和90%。结论成功构建了靶向YB-1基因的shRNA表达质粒,并筛选出1种对YB-1基因有显著抑制作用的shRNA,为进一步研究YB-1对乳腺癌细胞侵袭、迁移和多药耐药性的影响奠定了基础。

Objective To construct a shRNA plasmid targeting Y-box binding protein-1 （YB-1） gene and identify its inhibitory efficacy. Methods Three pairs of shRNA chains targeting YB-1 gene were designed and synthesized, then annealed to form double strand, and inserted into plasmid pGenesil-1 digested with restriction enzyme. The constructed three recombinant plasmids pGSi1, pGSi2 and pGSi3 were transfected to human breast cancer MDA-MB-231 cells in mediation of liposome and determined for transcription level of YB-1 mRNA by fluorescent quantitative PCR and for expression level of YB-1 protein by Western blot. Results Restriction analysis and sequencing proved that recombinant plasmids pGSi1, pGSi2 and pGSi3 were constructed correctly. Both the expressions of YB-1 gene at mRNA and protein levels in MDA-MB-231 cells transfected with the three plasmids decreased significantly. The inhibitory rates of pGSi2 on YB-1 gene expression at mRNA and protein levels were 57. 4% and 90% respectively, which were significantly higher than those of pGSi1 and pGSi3. Conclusion A shRNA plasmid targeting YB-1 gene was successfully constructed, and a shRNA with significantly inhibitory effect on YB-1 gene was screened, which laid a foundation of further study on the effects of YB-1 on invasion, migration and multidrug-resistance of breast cancer cells.