【摘要】 目的 构建特异性沉默Ku80基因的真核表达栽体,检测其对乳腺癌基因HER2表达的影响。方法 设计2条表达短发夹RNA的互补DNA序列,分别克隆至真核表达载体pGCsi-U6/Neo/DsRed构建shRNA-Ku80,转化大肠埃希菌DH5α菌株扩增后,提取质粒测序分析,以相同的方法构建并鉴定Ku80基因的真核表达载体pEGFP-N1-Ku80;将2种shRNA-Ku80分别与pEGFP-N1-Ku80共转染HEK293细胞株,通过流式细胞仪检测荧光强度筛选高效敲减靶点,并在基因和蛋白水平观察抑制Ku80对乳腺癌基因HER2表达的影响。结果 成功构建Ku80特异性shRNA表达载体,shRNA-Ku80对靶点Ku80敲减效率达到59.50%,抑制Ku80表达使乳腺癌基因HER2在基因和蛋白水平明显降低。结论成功构建shRNA-Ku80载体,在体外可有效抑制Ku80的表达,使癌基因HER2的表达明显下调,提示Ku80有望成为乳腺癌治疗的基因靶点。更多还原

【Abstract】 Objective To construct the eukaryotic expression vcctor to silencing Ku80 gene and explore the effects of silencing of Ku80 on the expression of HER2 in breast cancer cells.Methods Two pairs of Ku80 DNA sequences were designed according to the Ku80 sequence in GeneBank,the sequence was cloned into pGCsi-U6/Neo/DsRed and then sequence analysis was performed,and used the same method to construct and identify Ku80 eukaryotic expression vcctor pEGFP-Nl-Ku80.Recombinant plasmids of two pGCsi-U6/Neo/DsRed/shRNA-Ku80 and a pEGFP-Nl-Ku80 were cotransfected into HEK293 cell by Lipofectamine2000 respectivcly,the optimal vcctor of two shRNA was chosen by flow cytometry testing the fluorescence of pEGFP-Nl-Ku80 fusion protein,which RNAi inhibitory effect was further confirmed by RT-PCR and Western blot checking HER2 in the breast cancer cell line BT-474.Results The specific shRNA and Ku80 coding sequences were consistent with the designed fragments,the inhibtive effect of the optimal shRNA vcctor on the expression of Ku80 was up to 59.50%tested by flow cytometry,and the level of HER2 decreasd significantly examined by RT-PCR and Western blot fellowed by transfected with shRNA-Ku80.Conclusion The RNAi eukaryotic vcctor pGCsi-U6/Neo/DsRed/shRNA-Ku80 was successfully constructed,which could effectively inhabited the expression of Ku80,and the HER2 was downregulated at the level of mRNA and protein accordingly in breast cancer cells.更多还原