Shan Zhixin, Lin Qiuxiong, Deng Chunyu, Li Xiaohong, Huang Wei, Tan Honghong, Fu Yongheng, Yang Min, Yu Xi-Yong
Research Center of Medical Sciences, Guangdong Provincial People's Hospital, Guangzhou, 510080, China.
Mol Biol Rep. 2009 Jul;36(6):1483-9. doi: 10.1007/s11033-008-9339-8. Epub 2008 Aug 30.
Gene silencing can be mediated by small interfering RNA (siRNA) and microRNA (miRNA). To investigate the potential application of using a precursor microRNA (pre-miRNA) backbone for gene silencing, we studied the inhibition efficiency of exogenous GFP and endogenous GAPDH by conventional shRNA- and pre-miRNA-designed hairpins, respectively. In this study, the conventional shRNA-, pre-miRNA-30-, and pre-miRNA-155-designed hairpins targeting either GFP or GAPDH were transfected into the HEK293 cells that were mediated by the pSilencer-4.1-neo vector, which carries a modified RNA polymerase II-type CMV promoter. Comparisons with conventional GFP shRNA showed that GFP levels were reduced markedly by pre-miRNA-30- and pre-miRNA-155-designed GFP shRNAs by fluorescence microscopy. The consistent results from semi-quantitative RT-PCR and Western blot analysis revealed that pre-miRNA-30- and pre-miRNA-155-designed GFP shRNAs could suppress GFP expression significantly. As for endogenous GAPDH, the results from semi-quantitative RT-PCR and Western blot analysis showed that pre-miRNA-30- and pre-miRNA-155-designed GAPDH shRNAs could suppress GAPDH expression even more efficiently than conventional GAPDH shRNA. Together, this study confirmed the efficiency of gene silencing mediated by pre-miRNA-30- and pre-miRNA-155-designed shRNAs, demonstrating that pre-miRNA-designed hairpins are a good strategy for gene silencing.
基因沉默可由小分子干扰RNA（siRNA）和微小RNA（miRNA）介导。为了研究使用前体微小RNA（pre-miRNA）骨架进行基因沉默的潜在应用，我们分别研究了传统短发夹RNA（shRNA）和pre-miRNA设计的发夹对外源绿色荧光蛋白（GFP）和内源性甘油醛-3-磷酸脱氢酶（GAPDH）的抑制效率。在本研究中，将靶向GFP或GAPDH的传统shRNA、pre-miRNA-30和pre-miRNA-155设计的发夹转染到由携带修饰的RNA聚合酶II型巨细胞病毒（CMV）启动子的pSilencer-4.1-neo载体介导的人胚肾293（HEK293）细胞中。荧光显微镜检查显示，与传统GFP shRNA相比，pre-miRNA-30和pre-miRNA-155设计的GFP shRNA显著降低了GFP水平。半定量逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹分析的一致结果表明，pre-miRNA-30和pre-miRNA-155设计的GFP shRNA可显著抑制GFP表达。对于内源性GAPDH，半定量RT-PCR和蛋白质免疫印迹分析结果表明，pre-miRNA-30和pre-miRNA-155设计的GAPDH shRNA比传统GAPDH shRNA更有效地抑制GAPDH表达。总之，本研究证实了pre-miRNA-30和pre-miRNA-155设计的shRNA介导的基因沉默效率，表明pre-miRNA设计的发夹是一种有效的基因沉默策略。
