人前列腺癌PC3细胞Bloom解旋酶基因干扰载体的构建

Construction of RNAi Vectors for RNA Interference of Bloom Helicase Gene in Human Prostate Cancer PC3 Cells

通过RNAi技术干扰人前列腺癌PC3细胞Bloom(BLM)解旋酶基因的表达。实验前期根据BLM解旋酶基因序列设计并合成两对sh RNA,插入到CMV-cop GFP-T2A-Puro-H1-mcs载体,构建针对BLM解旋酶基因的重组干扰载体CMV-cop GFP-T2A-Puro-H1-mcs-BLM1、CMV-cop GFPT2A-Puro-H1-mcs-BLM2。经测序载体构建成功后,用脂质体将所构建的载体及阴性对照载体转染前列腺癌PC3细胞,通过荧光定量PCR和Western blot检测转染细胞的BLM解旋酶表达情况。检测结果显示,成功构建了BLM解旋酶RNAi真核表达载体;利用脂质体转染PC3细胞后,荧光定量PCR和Western blot鉴定结果表明,BLM解旋酶的表达水平显著降低,即Bloom RNAi真核表达载体在m RNA和蛋白质水平阻断了BLM解旋酶的表达。

RNA interference （RNAi） technology was used in this research for the sake of down-regulating the expression of Bloom helicase in human prostate cancer PC3 cells. At the early stage of the experiment, vectors capable of producing short hairpin RNA （shRNA） molecule for Bloom helicase were constructed using the mamma- lian expression plasmid vector CMV-copGFP-T2A-Puro-HI-mcs. After the vectors were successfully constructed and confirmed by DNA sequencing, the PC3 cells were transfected with the constructed positive and negative RNAi vectors. Real-time fluorescence quantitative PCR （RTFQ PCR） and Western blot were used to examine the mRNA and protein levels in the transfected PC3 cells. The results indicated that the Bloom helicase mRNA and protein level decreased significantly after tranfection of the positive vectors in PC3 cells, while in negative plasmid transfected PC3 cells, Bloom helicase expression remained the same level as the normal cells. These data suggested that Bloom helicase expression could be inhibited by shRNA transfectants in PC3 cells at mRNA and protein levels.