title: An efficient method to enhance gene silencing by using precursor microRNA designed small hairpin RNAs.
abstract: 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.
authors.affiliation: Research Center of Medical Sciences, Guangdong Provincial People's Hospital, Guangzhou, 510080, China.
authors.forename: Zhixin
authors.initials: Z
authors.lastname: Shan
authors.forename: Qiuxiong
authors.initials: Q
authors.lastname: Lin
authors.forename: Chunyu
authors.initials: C
authors.lastname: Deng
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Li
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
authors.forename: Honghong
authors.initials: H
authors.lastname: Tan
authors.forename: Yongheng
authors.initials: Y
authors.lastname: Fu
authors.forename: Min
authors.initials: M
authors.lastname: Yang
authors.forename: Xi-Yong
authors.initials: XY
authors.lastname: Yu