Figure 5.

SNHG17 Directly Binds to miR-214-3p and Acts as a Molecular Sponge in the Cytoplasm

(A) Representative FISH images show the distribution of SNHG17 in OVCAR-3 and PEO1 cells (red). The nuclei were stained by DAPI (blue). (B) Relative SNHG17 expression levels in the nuclear and cytoplasm fractions of OVCAR-3 and PEO1 cells. Nuclear controls, U6; cytosolic controls, GAPDH. (C) The relative expression of candidate microRNAs that could potentially bind to SNHG17 was quantified by qRT-PCR after the biotinylated-SNHG17 pull-down assays in OVCAR-3 cells. (D) miR-214-3p expression was detected in OVCAR-3 cells transfected with SNHG17 siRNAs or the SNHG17-overexpressing vector by qRT-PCR. (E) qRT-PCR analysis of miR-214-3p expression in 90 pairs of OC and corresponding adjacent normal tissues. (F) Dual-luciferase reporter assays of WT and mutant type (putative binding sites for miR-214-3p were mutated) SNHG17 luciferase report vectors. Lower panel: sequence alignment of miR-214-3p and their predicted binding sites (green) for SNHG17. Predicted microRNA target sequence (654 bp–665 bp and 902 bp–911 bp, blue) in SNHG17 (Luc-SNHG17-WT) and positions of the mutated nucleotides (red) in SNHG17 (Luc-SNHG17-mt). (G) RIP assays with an anti-Ago2 antibody to assess endogenous Ago2-binding RNAs; immunoglobulin G (IgG) was used as the NC. The levels of SNHG17 and miR-214-3p were determined by qRT-PCR and presented as the fold enrichment in Ago2 relative to the input. (H) The correlation between SNHG17 and miR-214-3p was analyzed in 90 paired OC samples (n = 90; r = −0.291, p < 0.001). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.