siRNA表达载体稳定沉默肺癌A549细胞MEKK3基因的细胞株建立

目的构建人MEKK3基因的小干扰RNA（siRNA）表达载体并建立稳定沉默MEKK3基因表达的肺癌A549细胞克隆株。方法设计并合成针对人MEKK3基因4个不同部位siRNA靶点的模板DNA序列，以BamHI及Hindm酶切位点分别克隆入pSilencer4．1-CMVhygro载体，测序鉴定后以脂质体分别转染入A549细胞，Western印迹检测它们对MEKK3表达的抑制，并选择抑制效率最高的表达载体转染入A549细胞，潮霉素筛选后获得含该载体的抗性细胞克隆株，荧光实时定量PCR检测该细胞克隆株中MEKK3表达抑制。结果测序鉴定表明4个MEKK3siRNA表达载体正确无误；Western印迹结果显示对MEKK3的表达有抑制作用，其中pSilencer4．1-MEKK3siRNA2的抑制率达84％；荧光实时定量PCR结果表明，pSilencer4．1-MEKK3siRNA2可稳定沉默MEKK3mRNA的表达，有效率达83％。结论成功地构建了针对人MEKK3基因的siRNA表达载体，并成功地建立了能高效且稳定地沉默MEKK3基因表达的肺癌A549细胞克隆株。

Establishment of a Lung Cancer A549 Cell Line Stably Silencing MEKK3 by Small Interfering RNA

Objective To construct a small interfering RNA （siRNA） expression vector targeting （Mitogen-activated protein kinase/extracellular signal-regulated protein kinase） kinase kinase3 （MEKK3） and establish a lung cancer A549 cell clones stably silencing MEKK3. Methods Four different siRNA template DNA sequences targeting on different sites of MEKK3 gene were designed by the application of Ambion＇s siRNA design software. These corresponding DNA fragments were synthesized in vitro, and were then cloned into the pSilencer 4. 1-CMV hygro vector with its two restrictive enzyme BamH Ⅰ and HindⅢ sites. The recombinant vectors were confirmed by DNA sequencing, and were then transfected into A549 cells with Lipofectamine2000. Western blot was eartied out to analyze the suppression effect of MEKK3 siRNA expression vectors and to screen the best vector that had the highest effect on inhibition of MEKK3 expression. Lung cancer A549 cell clones stably expressing siRNA against MEKK3 was established by hygromycin B screening, and its inter- ference effect was assessed by real-time fluorescence quantitative PCR. Results DNA sequencing confirmed that the MEKK3 siRNA expression vectors were successfully constructed, and the results of western blot showed that all of the four MEKKs siRNA expression vectors could effectively silence MEKK3 expression, and the best one was pSilencer4. 1-MEKK3 siRNA2 with a suppression ratio up to 84 %. Real time PCR data exhibited that pSilencer4. 1-MEKK3siRNA2 was able to stably si- lence the expression of MEKK3 with a suppression ratio up to 83 %. Conclusion The siRNA ex-pression vector targeted on MEKK3 was successfully constructed, and A549 stable clones with high effect MEKK3 silencing was also successfully established.