Rapid Generation of MicroRNA Sponges for MicroRNA Inhibition (original) (raw)
Figure 3
Bulged miRNA sponges can effectively inhibit miRNA function.
(A) GFP competition assay in WEHI-231 B-cell lymphoma cells infected with empty vector (EV), bulged and perfect miR-19 sponges. Bulged MBS sponges have mild (2 MBS) or strong effects (6 and 20 MBS) on cell growth while perfect MBS sponges and the empty vector control have no effect. Legend: x = EV; Δ = P-2MBS; □ = P-6MBS; ○ = P-18MBS; ▴ = B-2MBS; ▪ = = B-6MBS; ▾ = B-20MBS. (B) From the experiment shown in Fig. 3a, EV, perfect en bulged MBS sponge infected GFP+ cells were sorted at day 14. Quantification of sponge (LTR driven) and GFP transcript levels (PGK driven) by qRT-PCR revealed lowered sponge/GFP transcript ratios for the perfect sponges but not for the bulged sponges as compared to the empty vector control. The number of MBS per sponge vector is indicated on the x-axis. (C) Quantification of sponge transcript levels in the AGO2-immunoprecipitated (IP) fraction of miR-155 sponge infected KM-H2 cells. AGO2-IP of miR-155 sponge infected KM-H2 cells was performed and followed by quantification of miR-155 sponge transcript levels in total, flowthrough (FT) and IP fraction of AGO2 and IgG IP experiments. MiR-155 sponge transcripts are highly enriched in the AGO2-IP fraction but not in the IgG-IP control fraction. In the inset of the graph a Western Blot which shows that AGO2 levels are enriched in the IP fraction upon AGO2-IP but not in the IgG-IP control. (D) GFP competition assay in WEHI-231 cells infected with individual bulged miRNA sponges against each member of the miR-17∼92 cluster and combination (combi-sp) sponges against each miRNA family of the miR-17∼92 cluster. As a control, empty vector and a combi-sp vector with seed scrambled miRNA BS (combi-scr) were used. All individual miRNA sponges show negative effects on cell growth, however, the strongest effects are observed when all miRNAs of the miR-17∼92 cluster are inhibited simultaneously. No effects on proliferation were observed with the empty vector and combi-scr vector controls. Legend: x = EV; □ = miR-17-5p-sp (12MBS); Δ = miR-18a-sp (10MBS); * = miR-19-sp (20MBS); ı = miR-20a-sp (7MBS); ○ = miR-92a-sp (10MBS); ▪ = Combi-sp1 (4×3MBS); ▴ = Combi-sp2 (4×3MBS); • = Combi-scr (4×3MBSscr). * = p-value <0.05; ** = p-value <0.01; *** = p-value <0.001.