Fig. 4.

BCRC-3 sponges miR-182-5p, and overexpression of BCRC-3 reverses the down-regulation effect of miR-182-5p on p27. a The luciferase activity of p27 3’UTR and promoter after transfection with BCRC-3 overexpression vectors in EJ or T24 T cell lines. b The relative luciferase activities of p27 3’UTR and truncates in EJ or T24 T cells transfected with BCRC-3 overexpression vector. Schematic Sequence of p27 3’UTR full length and truncates (up panel). c & d The efficiency of BCRC-3 pull-down assay was verified by RT-PCR (c) or real-time PCR (d). GAPDH was used as negative control. Relative level of BCRC-3 was normalized to input. e qRT-PCR analysis of the expression of 7 candidates in the EJ and T24 T lysates after biotin-BCRC-3 pull-down assay. f RNA-FISH detection of BCRC-3 in T24 T cells. Nuclei were stained blue with DAPI. BCRC-3 was stained red with cy3. Locked nucleic acid miR-182-5p probes were labeled with Dig (scale bar, 10 μm). g qRT-PCR analysis of the expression levels of BCRC-3 or GAPDH in the EJ and T24 T lysates after biotin-miR-182-5p pull-down assay. GAPDH was used as negative control. h The luciferase activities of wild type p27 3’UTR and mutant p27 3’UTR after transfection with miR-182-5p mimic in EJ or T24 T cell lines. i & j qRT-PCR and western blot analysis of the expression levels of p27 in EJ or T24 T cells after co-transfection with BCRC-3 overexpression vector and miR-182-5p mimics. (Data are mean ± SEM of three experiments. Student’s t-test analyzed the difference in a-b, d-e, g-i. * P < 0.01 vs. vector, p27 3’UTR, oligo probe, biotin-miR-182-5p WT, or mimic NC. & P < 0.05 vs. mimic NC. # P < 0.05 vs. mimic NC + BCRC-3)