抑制Dicer基因对shRNA功能发挥的影响

本文将Dicer基因的RNA酶III结构域作为靶区,设计并构建了两个抗Dicer基因的小发夹样RNA(shRNA)表达载体,将其转染2215、结肠癌TC细胞和基因组中整合有绿色荧光蛋白基因(GFP)的HepG2A9细胞,通过RT-PCR评价RNA干扰抑制Dicer基因表达的效率;当HepG2A9细胞Dicer基因表达被上述RNA干扰抑制时,再转染抗GFP的shRNA表达载体,通过RT-PCR和荧光显微镜观察GFP表达水平。结果显示,在不同细胞系中,这两个抗Dicer基因shRNA表达载体,均能明显抑制Dicer基因的表达;当Dicer基因受抑时,后续转染抗GFP的shRNA表达载体不能有效抑制GFP的表达。结果表明,抗Dicer基因shRNA表达载体,能够明显抑制Dicer基因的表达;shRNA表达载体的功能发挥需要Dicer酶的直接参与。

Inhibition of Dicer Gene and Its Influence on the Function of shRNA

In order to determine the effect of silencing Dicer on shRNA-mediated RNA interference, we constructed two shRNA expression vectors that targeted the RNAse III domain of the Dicer gene. The shRNA vectors were transfected into 2215 cells, colon cancer TC cells and Green Fluorescent Protein (GFP)-tagged HepG2 A9 cells using lipofectamineTM 2000. The level of Dicer mRNA was assessed by RT-PCR. After the knocking-down of the Dicer gene was confirmed in the GFP-tagged HepG2 A9 cells, another shRNA expression vector that targeted the GFP gene was transfected in these cells. The suppression of GFP expression was evaluated by RT-PCR and fluorescence microscopy. We found that anti-Dicer shRNA expression vectors efficiently inhibited the expression of Dicer in the tested cells lines. Moveover, when the Dicer expression was silenced in GFP-tagged HepG2 A9 cells, the GFP-targeting shRNA expression vector was less effective in silencing GFP expression. Thus, our results indicated that the expression of Dicer could be inhibited by anti-Dicer shRNA expression vectors, and that the expression silencing activity of shRNA expression vectors is dependent on the presence of Dicer enzyme.