Fig. 5

Subcellular localization of HAGLROS and its “sponge” function as a ceRNA competing with miR-100-5p. a RNA was extracted from the nuclear and the cytoplasmic fractions of SGC-7901 and BGC-823 cells and HAGLROS expression of the nuclear and the cytoplasmic fraction was measured by qRT-PCR. GAPDH was used as a cytosolic marker, and U6 was used as a nuclear marker. b FISH was used to confirm HAGLROS location in SGC-7901 and BGC-823 cells, using Cy3 probes for HAGLROS, DAPI for nuclear staining. c miR-100-5p expression was examined in SGC-7901 and BGC-823 cells with HAGLROS knockdown by siRNAs, and HAGLROS expression was tested to determine the transfection efficiencies. d HAGLROS levels were examined in SGC-7901 and BGC-823 cells transfected with miR-100-5p, and miR-100-5p levels were tested for transfection efficiencies. e The expression of miR-100-5p in tumor samples of GC compared to adjacent non-cancerous tissues. f Wild-type or mutant HAGLROS plasmid was co-transfected with miR-NC or miR-100-5p mimics into 293T cells, and relative luciferase activities were measured to determine the level of interaction between miR-100-5p and HAGLROS. g RNA levels in immunoprecipitates are presented as fold enrichment relative to IgG in AGO₂ cells by RIP experiment. Error bars indicate the means ± S.E.M. *P < 0.05, **P < 0.01, #P < 0.05