Figure 5

ADAMTS9-AS1 acted as a ceRNA by sponging miR-6516-5p. (A) The subcellular localization of ADAMTS9-AS1 in EESCs and NESCs was analyzed using an RNA-FISH assay. Probes targeting ADAMTS9-AS1 were stained green, and DAPI was used to stain the nuclei of EESCs and NESCs. (B) Schematic representation of the predicted binding sites of miR-6516-5p in the ADAMTS9-AS1 sequence. (C) Luciferase activity was assessed using a dual-luciferase reporter assay in ESCs cotransfected with miR-6516-5p and recombinant luciferase reporter plasmids containing ADAMTS9-AS-wt (or ADAMTS9-AS-mut). Renilla luciferase was used as the internal control for normalizing transfection efficiency, and the data are shown as the relative ratio of firefly luciferase activity. (D) Luciferase activity was assessed using a dual-luciferase reporter assay in ESCs cotransfected with miR-6516-5p (or miR-6516-5p-mut or miRcont) and recombinant luciferase reporter plasmids containing ADAMTS9-AS-wt. (E) The direct combination of ADAMTS9-AS with miR-6516-5p was assessed through an RNA pull-down assay using 3′-biotinylated miR-6516-5p as bait. (F) Schematic representation of the predicted binding sites of miR-6516-5p in the GPX4-3’UTR sequence. (G) Luciferase activity was assessed using a dual-luciferase reporter assay in ESCs cotransfected with miR-6516-5p (or miRcont) and recombinant luciferase reporter plasmids containing GPX4-3’UTR-wt (or GPX4-3’UTR-mut). (H) The protein expression of GPX4 was assessed using western blot analysis in ESCs after miR-6516-5p overexpression. (I) Luciferase activity was assessed using a dual-luciferase reporter assay in ESCs cotransfected with miR-6516-5p and luciferase reporters containing GPX4-3’UTR, ADAMTS9-AS-wt, or ADAMTS9-AS-Mut. *p < 0.05, **p < 0.01.