Research Paper Volume 15, Issue 12 pp 5734—5750

Figure 6. SNHG3 promotes CSNK2A1 expression through interaction of miR-485-5p and HuR protein. (A) The relative expression of candidate microRNAs which could potentially bind to SNHG3 were quantified by qRT-PCR after the biotinylated-SNHG3 pulldown assays in MDA-MB-231 cells. (B) Dual luciferase reporter assays were conducted with wild-type and mutant-type (putative binding sites for miR-485-5p were mutated) luciferase report vectors of SNHG3 and miR-485-5p. Right panel, sequence alignment of miR-485-5p and its predicted binding sites (green) of SNHG3. Predicted microRNA target sequence (blue) in SNHG3 (Luc-SNHG3-wt) and positions of mutated nucleotides (red) in SNHG3 (Luc-SNHG3-mt). (C) Dual luciferase reporter assays were conducted with wild-type and mutant-type (putative binding sites for miR-485-5p were mutated) luciferase report vectors of CSNK2A1 3’UTR and miR-485-5p. (D) RNA immunoprecipitation with an anti-Ago2 antibody was used to assess endogenous Ago2 binding to RNA, IgG was used as the control. The levels of SNHG3 and miR-485-5p were determined by qRT–PCR and presented as fold enrichment in Ago2 relative to input. (E, F) MiR-485-5p expression was detected by qRT-PCR after SNHG3 was silenced or overexpressed. (G, H) CSNK2A1 expression was detected by qRT-PCR in MDA-MB-231 cells with indicated treatment. (I) Representative image of silver-stained PAGE gels showing separated proteins that were pulled down using biotin-labeled SNHG3. A red frame indicates HuR. (J, K) CSNK2A1 expression was detected by western blot in MDA-MB-231 cells with indicated treatment.