Is increasing the copy number of the microRNA precursor in a vector really helpful for further improving the level of mature microRNA in stable cells?

Abstract

We read with great interest the article by Xu et al.1 The authors used lentiviral vectors to overexpress microRNA 122 (miR-122). They constructed three different vectors carrying one, four, or eight copies of the miR-122 precursor. Finally, the authors generated stable cells possessing eight copies of the miR-122 precursor. This is an ingenious method for further improving the expression level of miR-122. However, we think that some problems need to be discussed. First, how do we determine that the stable HepG2 cells possess eight copies of the miR-122 precursor when the cells are infected with lentiviral vectors carrying multiple copies? Why do we not use lentiviral vectors carrying 10 or more copies to further improve the level of miR-122? Are there data from the experiment using a gradually increasing number of miR-122 copies in vectors to construct miR-122–overexpressed stable cells? Recently, in our experimentation, HepG2 cells were transfected with vectors carrying a single copy of the miR-122 precursor. By screening different single clones, we obtained several HepG2 cell clones that had large differences in the expression level of miR-122. We deduced that the miR-122 precursor sequence could be integrated into several sites of the genome simultaneously. Information about the copy number can help us to choose optimal stable cell lines because of the existence of copy loss in the genome. We think that a stable cell line with a high copy number should be chosen when there are similar expression levels in different cell lines. Second, primary microRNA must be cleaved into precursor microRNA (pre-miRNA) by the nuclear RNase III endonuclease Drosha. Then, the pre-miRNA is actively transported from the nucleus to the cytoplasm by ras-related nuclear protein–guanosine triphosphate and the export receptor exportin 5.2, 3 The processes of enzyme digestion and export depend on not only the transcript sequences but also their space structure.4 We suppose that when eight or more copies of the miR-122 hairpin were chained to express in one primary transcript, the processing or export of pre-miRNA was likely influenced by the changes in the conformational construct of the primary transcript or pre-miRNA. Therefore, we want to know how to determine the optimal copy number of a microRNA precursor in a vector. In many studies, microRNA-overexpressed stable cell lines have been obtained by the integration of the microRNA precursor sequence into the cell genome.5, 6 In most cases, a vector carrying a single-copy precursor sequence has been chosen.7, 8 If the aforementioned problems can be solved with certainty, this novel method will be adopted widely in microRNA and RNA interference research, and a better cell model will be used in RNA-related research. Fang Wang*, Fu Yang*, Ling Zhang*, Shuhan Sun*, * Department of Medical Genetics, Second Military Medical University, Shanghai, China