An efficient method to enhance gene silencing by using precursor microRNA designed small hairpin RNAs

作者：Shan, Zhixin; Lin, Qiuxiong; Deng, Chunyu; Li, Xiaohong; Huang, Wei; Tan, Honghong; Fu, Yongheng; Yang, Min; Yu, Xi-Yong^*

来源：Molecular Biology Reports, 2009, 36(6): 1483-1489.

DOI：10.1007/s11033-008-9339-8

• MicroRNAs
• RNA interference
• RNA Pol II promoter
• Small hairpin RNA

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.

• 出版日期2009-7
• 单位广东省人民医院; 广东省心血管病研究所