Fig 1.
Schematic outline for the construction of pLVX-dTuD vector.
(A) Overview of a dTuD lentivirus-based vector (pLVX-dTuD) encoding two TuD RNA molecules. Each TuD RNA includes one stem (18 nt), two MBS with bulge (4 nt), one stem-loop (10 nt) and four linkers (3 nt) between stem and MBS. (B) Strategy to construct pLVX-dTuD vector. Step 1: preparation of the MBS-recipient vector. Step 2: preparation of MBS-donor by two-step PCR. Step 3: MBS-donor fragment was inserted into MBS-recipient vector by utilizing the Golden Gate cloning.
Fig 2.
Dose-dependency of dTuD on the expression of related miRNAs.
293A cells were transfected with dTuD targeting miR-20a, -92a, -195 or -497 at the concentration of 0, 100, 200, 400 and 800 ng/mL. qRT-PCR was performed to evaluate the expression of miRNA. Data was shown as the percentage of miRNA in the cells transfected with dTuD via that in the cells transfected with dTuD-Ctrl. Data were represented as the mean ± SD (n = 3).
Fig 3.
Specificity of dTuD on related miRNAs.
(A) miR-15a, -15b, -16, -107, -195, -424, -497 and -503 sequences. Seed sequences are shown in box. (B-C) The expression levels of indicated miRNAs in the 293A cells transfected with dTuD-miR-195 (B) or dTuD-miR-497 (C) were evaluated 48h after the transfection by qRT-PCR. The expression of miRNA was normalized to that of dTuD-Ctrl transfected cells. Data were represented as the mean ± SD (n = 3). ** p < 0.01.
Fig 4.
The inhibitory effect on miR-223 activity by dTuD-miR-223.
(A) Schematic representation of 3′UTR luciferase reporter plasmid in which the 3′UTR of RhoB contained the binding sites of miR-233 (1726–1283 bp from 5′end) was fused to the 3′end of Firefly Luciferase (FLuc) after stop codon. (B) Dual-luciferase assay in 293A cells co-transfected with multiple plasmids as indicated. H1-TuD-miR-223 and U6-TuD-miR-223 represent the modified dTuD vector harboring a single TuD-miR-223 expression cassette driven by H1 and U6 promoter, respectively. After performing dual luciferase assay, the ratio FLuc/Renilla Luc (RLuc) was normalized to that of dTuD-Ctrl. (C) Expression level of miR-223 or (D) RhoB mRNA in 293A cells transfected with antagomir, TuD or dTuD against miR-233. Data were represented as the mean ± SD (n = 3). * p < 0.05, *** p < 0.001.
Fig 5.
Inhibitory effects of dTuDs on protein expression of miRNA target.
The protein expression level of CDC25A (A) or CCNE1 (B) in C2C12 cells transduced with lentivirus expressing dTuD-miR-322 or dTuD-miR-497, respectively. CDC25A and CCNE1 are validated target genes of miR-322 and -497, respectively. The protein expression level was normalized to that of dTuD-Ctrl. Data were represented as the mean ± SD (n = 3).* p < 0.05.
Fig 6.
Inhibitory effect of dTuD-miR-1 in mice.
(A) Schematic representation of dTuD-miR-1 intraperitoneal administration into mice. Two-week-old BALB/c mice were used, and received an intraperitoneal injection of dTuD every two weeks. Gastrocnemius muscles were collected two weeks after the third injection. (B) The expression of miR-1 in gastrocnemius muscle was measured by qRT-PCR. (C) The protein of myogenin, one of the miR-1 down-stream targets, was evaluated by Western blot. The expression level of miR-1 or myogenin was normalized to that of dTuD-Ctrl. Data were represented as the mean ± SD (n = 4) and compared between groups using the two-tailed Student’s t-test. ** p < 0.01, *** p < 0.001.
Fig 7.
High-throughput screening for miRNAs involved in AP-1 pathway by dTuDs library and AP-1 luciferase reporter system.
(A) Schematic representation of AP-1 indicator-sensor plasmid. (B-C) 293A cells were transfected with a mixture of three vectors: AP-1 luciferase reporter plasmid, RLuc plasmid and each dTuD vector. 24h after transfection, cells were treated with (black bars) or without (open bars) PMA for 18h and then harvested for luciferase activity assays. Relative activity of AP-1 were represented as the ratio of FLuc/RLuc normalized to that in cells transfected with dTuD-Ctrl. dTuDs targeting 88 miRNAs were tested for the first screen (B), and those miRNAs with a fold change > 2-fold were further verified following the same procedure as the first screen (C). The dashed lines indicate 2.0 fold above and 50% below the dTuD-Ctrl. Data were represented as the mean ± SD (n = 3). * p < 0.05, ** p < 0.01.