Figure 3. LOC146880 sponges miR-328-5p in ESCC cells. (A) FISH analysis shows localization of LOC146880 (green) in the cytoplasm and nucleus (blue) of Kyse30 and TE-1 cells. (B) Relative levels of LOC146880 in the nuclear and cytosolic fractions of Kyse30 and TE-1 cells. U6 and GAPDH were used as controls for the nuclear and cytosolic fractions. (C) RIP assay shows fold enrichment of LOC146880 and miR-328-5p in the anti-Ago2-antibody and IgG control groups. (D) Dual luciferase reporter assay results show relative luciferase activity of Kyse30 cells co-transfected with vector containing LOC146880-WT plus vectors containing one of the three miRNAs (miR-4715-3p, miR-328-5p and miR-4492). (E) Predicted miR-328-5p seed region in the wild-type (WT) and mutated (Mut) LOC146880. (F) Dual luciferase reporter assay shows relative luciferase activity in Kyse30 cells co-transfected with luciferase reporter plasmid containing wild type (WT) or mutant (Mut) LOC146880 and miR-328-5p mimics, miRNA-328-5p NC act as control. (G) TCGA database analysis shows expression levels of miR-328-5p in ESCC (N = 183) and adjacent normal esophageal tissues (N = 12). (H) QRT-PCR analysis shows expression levels of miR-328-5p in 21 pairs of ESCC and paracancerous esophageal tissues. (I) Spearman’s rank correlation analysis shows inverse relationship between LOC146880 and miR-328-5p expression levels in the 21 pairs of ESCC and paracancerous esophageal samples. (J) QRT-PCR analysis shows expression levels of miR-328-5p in control and LOC146880 silenced Kyse30 and TE-1 cells. *P < 0.05, **P < 0.01, ***P < 0.001.