Figure 2

Reciprocal stabilization of miR-1254 and CCAR1. (A-B) MCF-7 cells were transfected with negative control (NC), miR-1254 mimic (miR-1254), pcDNA3.1 (+) (vector) or miR-1254 sponge (sponge). The levels of miR-1254, CCAR1 and pri-CCAR1 (primary CCAR1 transcript) were determined by qPCR. Error bars indicate SEM. ^***P < 0.001, NS, not significant. (C) Western blot analysis of CCAR1 with forced expression or antagonism of miR-1254. (D) Half-life of CCAR1 mRNA determined after forced expression or repression of miR-1254 in the presence of actinomycin D. Error bars indicate SEM. ^***P< 0.001. (E) Schematic diagram of the C5U-LUC plasmid. (F-G) MCF-7 cells were co-transfected with miR-1254 and C5U-LUC or a single-site mutant (C5U-LUC-Mut 1, C5U-LUC-Mut 2) or a double-site mutant (C5U-LUC-Mut 1 and 2), and luciferase mRNA level (F) or luciferase activity (G) of FL/RL was determined. Error bars indicate SEM. ^***P < 0.001. (H) Alignment of miR-1254 and its binding sequences on C5U. (I) Schematic diagram of CEG plasmid. (J-K) EGFP RNA levels of MCF-7 CEG and MCF-7 mutCEG cells upon forced expression (J) or repression (K) of miR-1254. Error bars indicate SEM. ^***P< 0.001. (L) Half-life of EGFP mRNA determined in MCF-7 CEG cells upon forced expression or repression of miR-1254 in the presence of actinomycin D. Error bars indicate SEM. ^***P < 0.001. (M) Protein levels of EGFP and β-ACTIN were determined after transfection with different doses of miR-1254 mimic or sponge in MCF-7 CEG and MCF-7 mutCEG cells. (N-O) MCF-7 cells transfected with vector or CEG (N), or shRNA of non-specific sequence (shNS) or shCCAR1 (O) for 48 h; the levels of CCAR1 5' UTR, CCAR1, miR-1254, and pri-miR-1254 (primary miR-1254 transcript) were determined by qPCR. Error bars indicate SEM. ^***P < 0.001. (P) Half-life of miR-1254 upon forced expression or depletion of C5U in presence of actinomycin D. Error bars indicate SEM. ^***P < 0.001. Student's t-test. See also Supplementary information, Figure S2.