Akt shRNA载体的构建及其对结肠癌细胞株Lovo Akt基因表达的抑制作用

Construction of the expression vector for short-hairpin RNA-mediated Akt gene silencing in Lovo cells

目的构建靶向人Akt基因的shRNA真核表达载体,通过RNAi下调Akt基因在结肠癌Lovo细胞系中的表达,观察对结肠癌细胞生长的影响。方法将靶向人Akt基因的两条shRNA(Akt1-U6-1#;Akt1-U6-2#)表达序列克隆到入门载体pENTRTM/U6的黏性末端,测序鉴定后与目的表达载体Plentio/Block-iT DEST Vector进行LR位点特异性重组;分别将入门载体与目的表达载体转染入Lovo细胞,对细胞株行RT-PCR和Western blotting检测,观察siRNA对靶基因Akt的抑制效果。结果 DNA测序证实pENTRTM/U6-TF-shRNA入门载体和Plentio/Block-iT DEST目的表达载体为阳性克隆,转染Lovo细胞后,Akt基因在mRNA和蛋白水平的表达量受到明显抑制。结论成功构建了靶向人Akt的shRNA表达载体,且该载体在体外能有效抑制Lovo细胞Akt基因表达,为进一步研究Akt的功能和肿瘤的基因治疗奠定了实验基础。

Objective To construct a eukaryotic expression vector of short-hairpin RNA（shRNA） targeting human Akt gene and assess the effect of Akt gene silencing on the growth of colon cancer Lovo cells.Methods Two shRNAs targeting human Akt gene were cloned into pENTRTM/U6 plasmid to obtain the entry clones,and the positive clones were verified by sequencing.After recombination of the pENTRTM/U6 entry constructs and Plenti6/Block-iT DEST vector,the positive clones were confirmed by sequencing.Lovo cells were transfected by the entry vector and DEST Vector,and RT-PCR and Western blotting were performed to detect the interference of Akt gene expressions.Results The pENTRTM/U6 entry clones carrying Akt shRNA and pLenti6/DEST-pENTRTM/U6-Akt shRNA were successfully constructed.Both of the vectors were transfected into Lovo cells and resulted in obvious knockdown of the mRNA and protein expressions of Akt.Conclusion The Akt siRNA expression vector constructed can significantly inhibit Akt gene expression in Lovo cells,which facilitates further studies of Akt function and tumor gene therapy.