RNA干扰DNA修复蛋白Ku80对乳腺癌基因HER2表达的影响  

Effects of RNAi-Mediated Gene Silencing of Ku80 on the Expression of HER2 in Breast Cancer Cells

作　　者：王萍 杨勤[2] 官全生[2] 

机构地区：[1] 解放军422医院检验科,广东湛江524009 [2] 广东医学院附属医院血液内科,广东湛江524001

出　　处：《现代检验医学杂志》2014年第2期44-47,51,共5页Journal of Modern Laboratory Medicine

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,shR-NA-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.Objective To construct the eukaryotie expression vector to silencing Ku80 gene and explore the effects of silencingof Ku80 on the expression of HER2 in breast cancer cells. Methods Two pairs of Ku80 DNA sequences were designed ac-cording to the Ku80 sequence in GeneBank,the sequence was cloned into pGCsi-U6/Neo/DsRed and then sequence analysiswas performed,and used the same method to construct and identify Ku80 eukaryotic expression vector pEGFP-N1-Ku80.Recombinant plasmids of two pGCsi-U6/Neo/DsRed/shRNA-Ku80 and a pEGFP-NI Ku80 were cotransfected intoHEK293 cell by Lipofectamine2000 respectively,the optimal vector of two shRNA was chosen by flow cytometry testing thefluorescence of pEGFP-N1-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 wereconsistent with the designed fragments,the inhibtive effect of the optimal shRNA vector on the expression of Ku80 was upto 59.50% tested by flow cytometry,and the level of HER2 deereasd significantly examined by RT-PCR and Western blotfellowed by transfeeted with shRNA Ku80. Conclusion The RNAi eukaryotic vector pGCsi-U6/Neo/DsRed/shRNA Ku80was successfully constructed,which could effectively inhabited the expression of Ku80,and the HER2 was downregulated atthe level of mRNA and protein accordingly in breast cancer cells.

关 键 词：DNA修复蛋白Ku80 人类表皮生长因子受体2 乳腺癌 

分 类 号：R737.943[医药卫生—肿瘤]