Transfection of microRNA Mimics Should Be Used with Caution.
Transfection of microRNA Mimics Should Be Used with Caution.
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DOI：
10.3389/fgene.2015.00340
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发表时间：
2015
影响因子：
3.7
通讯作者：
Xiao C
中科院分区：
文献类型：
--
作者：
Jin HY;Gonzalez-Martin A;Miletic AV;Lai M;Knight S;Sabouri-Ghomi M;Head SR;Macauley MS;Rickert RC;Xiao C
化学合成的microRNA（miRNA）模拟物的瞬时转染被广泛用于研究内源性miRNA的功能和机制。然而，目前尚不清楚转染的miRNA是否与内源性miRNA表现相似。在这里，我们表明，通过常用的方法将miRNA模拟物瞬时转染到HeLa细胞中导致高分子量RNA种类的积累和成熟miRNA水平的数百倍增加。相反，通过慢病毒感染或质粒转染HeLa细胞表达相同的miRNA，在原代淋巴细胞中转基因表达，以及在淋巴瘤和白血病细胞系中内源性过表达，并不导致高分子量RNA的出现。这些细胞中成熟miRNA水平的增加低于10倍，这足以抑制靶基因表达并驱动小鼠淋巴瘤的发展。此外，高浓度的miRNA模拟物的瞬时转染引起基因表达的非特异性改变，而在低浓度下达到与其他方法相当的表达水平，但未能有效抑制靶基因表达。小RNA深度测序分析显示，miRNA模拟物的引导链经常发生突变，而一些miRNA模拟物的非天然乘客链积累到高水平。高分子量RNA种类是由多联体化、miRNA模拟物的5′-和3′-末端加尾产生的几类RNA种类的异质混合物。我们推测，超生理水平的成熟miRNA和这些人造RNA种类导致基因表达的非特异性变化。我们的研究结果对设计和解释主要采用瞬时转染miRNA模拟物的实验具有重要意义。
Transient transfection of chemically synthesized microRNA (miRNA) mimics is being used extensively to study the functions and mechanisms of endogenous miRNAs. However, it remains unclear whether transfected miRNAs behave similarly to endogenous miRNAs. Here we show that transient transfection of miRNA mimics into HeLa cells by a commonly used method led to the accumulation of high molecular weight RNA species and a few hundred fold increase in mature miRNA levels. In contrast, expression of the same miRNAs through lentiviral infection or plasmid transfection of HeLa cells, transgenic expression in primary lymphocytes, and endogenous overexpression in lymphoma and leukemia cell lines did not lead to the appearance of high molecular weight RNA species. The increase of mature miRNA levels in these cells was below 10-fold, which was sufficient to suppress target gene expression and to drive lymphoma development in mice. Moreover, transient transfection of miRNA mimics at high concentrations caused non-specific alterations in gene expression, while at low concentrations achieved expression levels comparable to other methods but failed to efficiently suppress target gene expression. Small RNA deep sequencing analysis revealed that the guide strands of miRNA mimics were frequently mutated, while unnatural passenger strands of some miRNA mimics accumulated to high levels. The high molecular weight RNA species were a heterogeneous mixture of several classes of RNA species generated by concatemerization, 5′- and 3′-end tailing of miRNA mimics. We speculate that the supraphysiological levels of mature miRNAs and these artifactual RNA species led to non-specific changes in gene expression. Our results have important implications for the design and interpretation of experiments primarily employing transient transfection of miRNA mimics.