Figure 4

Rational design and characterization of miRancer. (A) Alignment between miR-1254, miRancer-1254, seed region match sequence mutant of miRancer-1254 (miRancer-1254 mut) and linear miR-1254-binding sequence (linear MBS). (B-D) miR-1254 levels (B), RL/FL of miR-1254 reporter (C) and NCOA1 mRNA levels (D) upon transfection with pSilencer 4.1-CMV (Vec), miRancer-1254, miRancer-1254 mut or linear MBS. Error bars indicate SEM. ^*P< 0.05, ^**P< 0.01, ^***P< 0.001, N.S., not significant. (E) Alignment between miR-7, miRancer-7, miR-7 sponge, miR-9, miRancer-9 and miR-9 sponge. (F) miR-7 and miR-9 levels by qPCR after transfection with pSilencer 4.1-CMV (Vec), miRancer-7, miRancer-9 for 48h. Error bars indicate SEM. ^***P< 0.001. (G) RL/FL of LUC-PTK2 3′ UTR or LUC-CDH1 3′ UTR after co-transfection with Vec, miRancer-7 or miRancer-9. Error bars indicate SEM. ^*P< 0.05, ^**P< 0.01. (H) miR-7 and miR-9 levels by qPCR after transfection with Vec, miR-7 sponge or miR-9 sponge. Error bars indicate SEM. ^**P< 0.01. (I) RL/FL of LUC-PTK2 3′ UTR or LUC-CDH1 3′ UTR after co-transfection with Vec, miR-7 sponge or miR-9 sponge. Error bars indicate SEM. ^*P< 0.05. (J-K) Ago2 depletion abrogates miRancer-7- or miRancer-9-mediated upregulation of miR-7 or miR-9. Error bars indicate SEM. ^***P< 0.001. (L) Schematic diagram of the miRancer-7-LUC, miR-7 sponge-LUC, miRancer-9-LUC, miR-9 sponge-LUC plasmid. (M-N) mRNA levels (M) or luciferase activity (N) of FL/RL of miRancer-7-LUC, miR-7 sponge-LUC, miRancer-9-LUC, miR-9 sponge-LUC upon miR-7 or miR-9 overexpression. Error bars indicate SEM. ^*P< 0.05, ^**P< 0.01, ^***P< 0.001. Student's t-test.