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目的构建针对Rictor基因的特异性RNA干扰质粒,稳定转染人膀胱癌T24细胞,观察其对目的基因Rictor表达的影响,为探讨沉默Rictor基因对人膀胱癌细胞生物学行为的影响奠定基础。方法根据GENEBANK提供Rictor基因系列,设计并化学合成三对Rictor-RNAi和一对阴性对照寡核苷酸序列,定向克隆入真核质粒载体pGCSIL-PUR中,构建pGCSIL-RICTOR-RNAi表达载体。各质粒载体瞬时转染到T24细胞,Western-Blot检测筛选载体。筛选出的载体稳定转染,Realtime-PCR和Western-Blot筛选干扰效果明显的稳定株。结果重组质粒pGCSIL-RICTOR-RNAi经酶切鉴定及DNA测序证实序列完全正确,成功构建Rictor-RNAi载体。瞬时转染T24细胞,RICTOR蛋白表达明显降低。稳定转染T24细胞,Rictor基因表达在mRNA水平和蛋白水平均明显降低。结论成功构建Rictor-RNAi表达载体,转染T24细胞后可有效抑制Rictor表达。
Objective To construct a specific RNAi plasmid targeting Rictor gene and stably transfected it into human bladder cancer T24 cells to observe its effect on the expression of target gene Rictor and lay a foundation for exploring the effect of silencing Rictor gene on the biological behavior of human bladder cancer cells. Methods According to the Rictor gene series provided by GENEBANK, three pairs of Rictor-RNAi and a pair of negative control oligonucleotide sequences were designed and synthesized. The pGCSIL-RICTOR-RNAi expression vector was constructed by direct cloning into the eukaryotic plasmid pGCSIL-PUR. Each plasmid vector was transiently transfected into T24 cells, and the vector was screened by Western-Blot. The selected vectors were stably transfected and the stable strains with obvious interference effect were screened by Realtime-PCR and Western-Blot. Results The recombinant plasmid pGCSIL-RICTOR-RNAi was verified by restriction enzyme digestion and DNA sequencing to confirm the correct sequence. Rictor-RNAi vector was successfully constructed. Transient transfection of T24 cells, RICTOR protein expression was significantly reduced. Stably transfected T24 cells, Rictor gene expression at the mRNA and protein levels were significantly lower. Conclusion The Rictor-RNAi expression vector was successfully constructed and transfected into T24 cells effectively inhibited Rictor expression.