Fig. 5

TINCR suppresses HER-2 expression by sponging miR-125b. a The expression level of TINCR in nuclear and cytoplasm of breast cancer cells was measured by qPCR. U1 (nuclear retained) and GAPDH (exported to cytoplasm) were used as controls. b FISH analysis of the subcellular location of TINCR with specific probe in breast cancer cells (Images were magnified at 20×). c RIP experiments were performed using the Ago2 antibody, and specific primers were used to detect the enrichment of TINCR and HER-2. d RIP measurement of the enrichment of Ago2 at TINCR and HER-2 transcripts relative to IgG in breast cancer cells infected with sh-TINCR. ^**P < 0.01 compared to sh-NC. e The putative sequences of miR-125b and TINCR with four binding sites (upper panel), miR-125b and HER-2 with one binding site (lower panel). f miR-125b expression was measured by qPCR in cells silenced with TINCR. ^*P < 0.05 compared to sh-NC. g Knockdown of miR-125b significantly rescued the suppressed HER-2 transcript (ERBB2) induced by TINCR knockdown, ^*P < 0.05. h-j Firefly luciferase activity normalized to Renilla luciferase activity (FL/RL) in breast cancer cells co-transfected with luciferase reporters with wild type transcripts of TINCR (h), E2F2 (i), and mutant type transcript E2F2 (j). miR-125b was overexpressed by miR-125b mimics to test the influence on luciferase activity. k RIP was performed using a HER-2 antibody to immunoprecipitate RNA and a primer to detect miR-125b. ^*P < 0.05 compared to sh-NC