Research Paper

Figure 5. LINC01224 acts as the molecular sponge of miR-193a-5p in gastric cancer cells. (A) The subcellular location of LINC01224 (red) in AGS and SNU-1 was detected by FISH. The nuclei were stained with DAPI (blue). Scale bar = 10 μm. (B) The relative expression level of LINC01224 in the nucleus and cytoplasm of AGS and SNU-1 cells. Nuclear control: U6; cytoplasmic control: GAPDH. (C) Venn diagram of overlapping target miRNAs predicted using the DIANA and starBase online analysis tools. (D) Dual-luciferase reporter assay was used to determine the direct targeting miRNAs of LINC01224. (E) Dual-luciferase reporter assays were performed with wild-type and mutant-type luciferase reporter vectors (mutations occurring at sites that may bind to miR-193a-5p). (F) Effects of overexpression and inhibition of miR-193a-5p on mRNA expression of LINC01224. (G) LINC01224 and miR-193a-5p were highly enriched in samples pulled down by biotinylated miR-193a-5p rather than control probes. (H) RNA immunoprecipitation with anti-Ago2 antibody was used to detect the binding of LINC01224 and miR-193a-5p to endogenous Ago2, and IgG was used as a control. LINC01224 and miR-193a-5p levels were determined by qRT-PCR and expressed as fold enrichment relative to the input Ago2. RIP efficiency of Ago2 protein was detected by Western blot.