Figure 3

Mechanism of miR-1254 and CCAR1 interaction in association with Ago2. (A) MCF-7 cells were transfected with shRNA of non-specific sequence (shNS), pooled shRNAs for Ago1 (shAgo1) or Ago2 (shAgo2). RNA levels of Ago1, Ago2, miR-1254 and CCAR1 were determined by qPCR. Error bars indicate SEM. ^***P < 0.001. (B-C) Ago2 depletion abrogates miR-1254's effect in promoting the expression (B) and half-life (C) of CCAR1. Error bars indicate SEM. ^***P < 0.001. (D-E) Ago2 depletion abrogates CCAR1 5' UTR-mediated increased expression (D) and half-life (E) of miR-1254. Error bars indicate SEM. ^***P < 0.001. (F-G) RNA immunoprecipitation of Ago2 or rabbit IgG in MCF-7 CEG cells with forced expression of miR-1254 (F), or in MCF-7 cells with forced expression of CCAR1 5′ UTR (G). EGFP and miR-1254 were detected by qRT-PCR and measured with input. Error bars indicate SEM. ^**P< 0.01. (H-I) Ago2 depletion abrogates CCAR1 enrichment by biotin-tagged miR-1254 (H) and miR-1254 enrichment by biotin-tagged probes against CCAR1 5′ UTR (I). Error bars indicate SEM. ^*P< 0.05, ^*P< 0.01. (J) Secondary structure of CCAR1 5′ UTR predicted by RNA fold. miR-1254-binding sites are indicated by arrows. (K) Sequence alignment of miR-1254 and structured site1 (SS1), linear site 1 (LS1), structured site 2 (SS2) and linear site 2 (LS2). (L-M) miR-1254 levels (L) or RL/FL of miR-1254 reporter (M) were determined upon transfection with pSilencer vector (Vec) or expression plasmids for CCAR1 5′ UTR (C5U), SS1, LS1, SS2, LS2. Error bars indicate SEM. ^*P< 0.05, ^**P< 0.01, ^***P< 0.001. (N) Schematic diagram of the SS1, LS1, SS2, LS2-LUC plasmid. (O-P) MCF-7 cells were co-transfected with miR-1254 and SS1, LS1, SS2 or LS2-LUC, and luciferase mRNA level (O) or luciferase activity (P) of FL/RL was determined. Dotted lines indicate the control level. Error bars indicate SEM. ^*P< 0.05. Student's t-test except in F-I and G, where χ-square test is used. See also Supplementary information, Figure S3.