Research Paper Volume 11, Issue 21 pp 9689—9708

Figure 5. circGRAMD1B acts as a molecular sponge for miR-130a-3p in GC cells. (A) Colocalization of circGRAMD1B and miR-130a-3p was measured using FISH in MGC803 and AGS cells. (B) Schematic illustration of circGRAMD1B-WT and circGRAMD1B-MUT luciferase reporter vectors. (C) A luciferase reporter assay was performed to detect the activities of circGRAMD1B -WT and -MUT in HEK-293T cells cotransfected with miR-130a-3p mimics or the miR-NC. (D) Screenshot of “Circular RNA Interactome” for has_circ_0004798 (circGRAMD1B), showing RBPs binding in different regions of this circRNA. (E–F) Anti-AGO2 RIP was performed in AGS cells after transfection with the miR-130a-3p mimics or miR-NC, followed by western blot and qRT-PCR analyses to detect AGO2 protein, circGRAMD1B, and miR-130a-3p. (G) RNA pull-down assays were performed in AGS cells, followed by qRT-PCR to detect the enrichment of circGRAMD1B and miR-130a-3p. (H) Relative expression level of miR-130a-3p was upregulated in GC tissues compared with paired noncancerous tissues according to qRT-PCR analysis. (I) Pearson correlation analysis of circGRAMD1B and miR-130a-3p expression in 32 GC tissues. (Values are shown as the mean ± standard error of the mean based on three independent experiments. *P < 0.05, **P < 0.01).