A quick and efficient approach for gene silencing by using triple putative microRNA-based short hairpin RNAs
A quick and efficient approach for gene silencing by using triple putative microRNA-based short hairpin RNAs
复制标题
使用三重推定的基于 microRNA 的短发夹 RNA 进行基因沉默的快速有效方法
DOI：
10.1007/s11010-008-9966-3
复制
发表时间：
2009-03-01
影响因子：
4.3
通讯作者：
Yu, X. Y.
中科院分区：
文献类型：
--
作者：
Shan, Z. X.;Lin, Q. X.;Yu, X. Y.
RNA干扰技术在基因功能研究中得到了广泛的应用。由于单个基因可以被多个sirna不同程度地抑制，因此通常通过敲除目标基因来筛选有效的sirna。基于mirna的短发夹RNA （short hairpin RNA, shRNA）是一种天然的RNAi诱导剂，已被用于siRNA的表达策略。我们研究了多种基于microrna的shRNA在基因沉默中的潜在应用，并研究了三个基于人mir155的shRNA表达载体对外源GFP和萤火虫荧光素酶（luc）的抑制效率。基于在线预测程序，共选择了三个针对gfporlucu的候选siRNA序列。以RNA聚合酶ii型巨细胞病毒（CMV）启动子为载体，用pcdna3载体介导的HEK293细胞转染靶向gfporluci的单和三联mirna -155 shrna。与阴性对照shrna比较，荧光显微镜显示，基于mirna -155的三重gfpshrna显著降低了GFP水平。双荧光素酶检测和实时定量RT-PCR结果一致显示，基于mirna -155的三组gfpshrna显著抑制dgfp表达（P< 0.01），与最有效的基于mirna -155的单组gfpshrna无显著差异（P< 0.05）。双荧光素酶检测和实时定量rt - pcr结果显示,三重microrna - 155 basedlucshrna显著suppressedlucexpression作为最有效的单一microrna - 155 basedlucshrna (P < 0.05)。这些研究证实了基于mirna -155的三种shrna介导的基因沉默效率。这表明多种基于mirna的shrna是一种快速而有价值的基因沉默策略。
The RNA interference (RNAi) technique has been widely used in gene function studies. It is typical to screen for effective siRNAs by knocking down targeted genes since a single gene can be suppressed by several siRNAs to varying degrees. The miRNA-based short hairpin RNA (shRNA) is a natural inducer of RNAi and has been used in siRNA expression strategies. We investigated the potential application of multiple putative microRNA-based shRNAs for gene silencing and studied the inhibition efficiency of exogenous GFP and firefly luciferase (luc) by triple human mir155-based shRNA expression vectors. A total of three candidate siRNA sequences targeted againstGFPorlucwere selected based on an online prediction program. Single and triple miRNA-155-based shRNAs targeted againstGFPorlucwere transfected into HEK293 cells mediated by the pcDNA3vector with an RNA polymerase II-type CMV (cytomegalovirus) promoter. Comparisons with negative control shRNAs revealed that GFP levels were markedly reduced by the triple miRNA-155-basedGFPshRNA by fluorescent microscopy. Consistent results from the dual luciferase assay and real-time quantitative RT-PCR revealed that the triple miRNA-155-basedGFPshRNA significantly suppressedGFPexpression (P< 0.01), without significant differences from the most effective single miRNA-155-basedGFPshRNA (P> 0.05). Results from the dual luciferase assay and real-time quantitative RT-PCR revealed that the triple miRNA-155-basedlucshRNA significantly suppressedlucexpression as the most effective single miRNA-155-basedlucshRNA (P< 0.05). These studies demonstrated the gene silencing efficiency mediated by the triple putative miRNA-155-based shRNAs. This suggested that multiple miRNA-based shRNAs are quick and valuable strategies for gene silencing.