Construction and Optimization of shRNA/siRNA Lentivral Vectors for microRNA-486-5p Using miR-30/451 and miRNA Sponges

By analyzing the hairpin structure of miR-486, it was found that the mature sequences of miR-486-5p and miR-486-3p are complementary. For this reason, it is probably the greatest challenge to express miR-486-5p using a lentiviral expression vector in C2C12 cells. Thus, we constructed shRNA/siRNA lentiviral expression vectors for microRNA-486-5p based on miR-30/451 and miRNA sponges. Oligonucleotide sequences (miR-486-5p TuD (tough decoy RNAs), miR-486-5p sponge, miR-486-5p-shRNA30 and miR-486-5p-shRNA451) were digested and cloned into the PLV-U6-MCS vector to generate U6-driven expression cassettes for mmu-miR-486-5p, and these vectors, including anti-miR-486-5p TuD, anti-miR-486-5p sponge, miR-486-5p-shRNA30 and miR-486-5p-shRNA451, were assessed by sequencing. The lentivirus vector was packaged in 293T cells according to the manufacturer’s instructions. Subsequently, C2C12 cells were infected using a lentivirus for the expression of miR-486-5p, and this lentivirus of PLV-U6-MCS served as a control. qRT-PCR analysis showed that anti-miR-486-5p TuD and the anti-miR-486-5p sponge decreased the expression level of miR-486-5p in C2C12 cells by 0.5-fold and 0.7-fold, respectively. Additionally, miR-486-5p-shRNA30 and miR-486-5p-shRNA451 upregulated the expression level of miR-486-5p in C2C12 cells by 15-fold and 3-fold, respectively. However, the expression level of miR-486-3p was increased up to 1000-fold in C2C12 cells containing the miR-486-5p-shRNA30 lentivirus, while other lentiviruses only led to 3-fold increases compared with the miR-486–5p-shRNA30 lentivirus in C2C12 cells. Additionally, western blot and gray density analysis demonstrated that the expression levels of AKT, and pAKT, ERK1/2 and pERK1/2, and Snail were upregulated (approximately 1.20-fold to 1.33-fold and 1.05-fold to 1.20-fold, respectively) in C2C12 cells infected with the anti-miR-486-5p TuD lentivirus and anti-miR-486-5p sponge lentivirus and downregulated (approximately 0.63–0.96 times and 0.72–0.92 times, respectively) in C2C12 cells infected with the miR-486-5p-shRNA30 lentivirus and miR-486-5p-shRNA451 lentivirus. In summary, anti-miR-486-5p TuD, which dramatically reduced the expression level of miR-486-5p and upregulated endogenous protein levels in C2C12 cells, was superior to the anti-miR-486-5p sponge in terms of interference expression of miR-486-5p. miR-486-5p-shRNA451, which did not lead to substantial expression of miR-486-3p and remarkably downregulated endogenous protein levels in C2C12 cells, was better than miR-486-5p-shRNA30 for overexpression of miR-486-5p. These results suggested that anti-miR-486-5p TuD and miR-486-5p-shRNA451 provide a simple way to build long-term effective overexpression or interference of miR-486-5p, to study miRNA function in C2C12 and mice, and to create a potential diagnostic method based on materials for diseases caused by miRNA deregulation.