Figure 5. CASC21 acts as a sponge for miR-539-5p in the cytoplasm. (A) Representative FISH images showed the location of CASC21 in HCT-116 and HCT-8 cells (red). Nuclei were stained by DAPI (blue). (B) Relative CASC21 expression levels in the nuclear and cytoplasm fractions of HCT-116 and HCT-8 cells. Nuclear controls: U6, cytosolic controls: GAPDH. (C) The relative expression of candidate microRNAs which could potentially bind to CASC21 were quantified by qRT-PCR after the biotinylated-CASC21 pull-down assays in HCT-116 cells. (D) MiR-539-5p expression was detected in HCT-116 transfected with CASC21 siRNAs or CASC21 overexpression vector by qRT-PCR. (E) Dual luciferase reporter assays of wild type and mutant type (putative binding sites for miR-539-5p were mutated) CASC21 luciferase report vectors. Up panel, sequence alignment of miR-539-5p and their predicted binding sites (green) of CASC21. Predicted micorRNA target sequence (blue) in CASC21 (Luc-CASC21-wt) and positions of mutated nucleotides (red) in CASC21 (Luc-CASC21-mt). (F) RIP assays with an anti-Ago2 antibody to assess endogenous Ago2 binding RNAs, IgG was used as the negative control. The levels of CASC21 and miR-539-5p were determined by qRT-PCR and presented as fold enrichment in Ago2 relative to input. (G) Left panel: expression of miR-539-5p in CRC generated from RNA sequencing data from TCGA database. Right panel: qRT-PCR analysis of miR-539-5p expression in 80 pairs of CRC and corresponding adjacent normal tissues. All data represent mean ± SEM (n = 3-6). *P < 0.05, **P < 0.01 and ***P < 0.001.