YB-1沉默点突变载体对YB-1基因敲除表型的回复

Rescue of the YB-1 knockdown phenotype with a mutanttype YB-1 vector

目的 Y盒结合蛋白-1(YB-1)在多种肿瘤中表达,并在转录和翻译水平调控相关癌基因中表达。为了进一步研究肺鳞癌细胞中YB-1的功能,构建了4个YB-1短发夹RNA(shRNA)干扰质粒pYr-1.1-YB-1-shRNA及YB-1沉默点突变质粒pYr-ads-1-YB-1 M。方法 pYr-1.1-YB-1-shRNA瞬时转染NIH293细胞,RT-PCR检测YB-1mRNA,筛选抑制效率最高的质粒,并稳定转染人肺鳞癌细胞株SK-MES-1,Western blot检测YB-1及PARP蛋白,免疫荧光检测核酸内切酶G(Endo G)亚细胞定位。针对干扰效率最高的靶序列,应用点突变PCR在YB-1基因表达质粒pYr-ads-1-YB-1上构建包含靶序列两个沉默点突变的质粒pYr-ads-1-YB-1 M。将pYr-1.1-YB-1-shRNA与pYr-ads-1-YB-1或pYr-ads-1-YB-1 M共转染SK-MES-1细胞,Western blot检测YB-1及PARP蛋白的表达。结果 4个pYr-1.1-YB-1-shRNA中2个干扰效率在60%以上,最高抑制率为70.3%;pYr-1.1-YB-1-shRNA质粒稳定转染SK-MES-1细胞能有效抑制YB-1蛋白的表达,并促进PARP蛋白的剪切及Endo G蛋白易位入核;pYr-ads-1-YB-1 M能回复pYr-1.1-YB-1-shRNA质粒所致的YB-1蛋白表达抑制,PARP蛋白剪切。结论 pYr-1.1-YB-1-shRNA特异性地通过抑制SK-MES-1细胞中YB-1的表达诱导细胞凋亡。

[Objective] Y-box binding protein-1 （YB-1） is a transcription/translation factor and become widely appreciated for its role in cancer. To further explore the function of YB-1, four plasmids pYr-1.1-YB-1-shRNA carrying short hairpin RNA （shRNA） targeting YB-1 and a mutant-type YB-1 plasmid pYr-ads-1-YB-1 M were constructed. [ Methods ] pYr-1.1-YB-1-shRNA were transiently transfected to NIH293 cells. RT-PCR was used to determine YB-1 mRNA and the most effective plasmid was chosen to stably transfected to SK-MES-1 cells, YB-1 and PARP proteins were examined by western blot, meanwhile the subcellular location of endonuclease G （Endo G） was observed by indirect immunofluorescence analysis; pYr-ads-1-YB-1 M, which contains two silent mutations in the region that is targeted by the most effective pYr-I.I-YB-I-shRNA, was created using point mutation PCR and pYr-ads-1-YB-1, pYr-I.I-YB-I-shRNA were co-transfected with either pYr-ads-1-YB-1 or pYr-ads-1-YB-1 M to SK-MES-1 cells, YB-1 and PARP were examined by Western blot. [ Results ] Of the four plasmids, two cut the amount of YB-1 by 60% in NIH 293 cells with the maximal suppression rate arriving at 70.3%. pYr-1.1-YB-1- shRNA could also significantly reduce YB-1 protein in SK-MES-1 cells, and meanwhile induce PARP cleavage and the release of Endo G from mitochondria to nucleus. Importantly both the knock down of YB-1 and the cleavage of PARP seen with pYr-1.1-YB-1-shRNA were rescued by the expression of the mutant type YB-1. [ Conclusion] pYr-1.1-YB-1-shRNA could induce apoptosis in SK-MES-1 cells through specifically knocking down YB-1.