Figure 4. RHPN1-AS1 acts as a ceRNA to sponge miR-596. (A) qRT-PCR analysis of RHPN1-AS1 expression in HEY, UWB 1.289, and ES-2 cells. (B) RNA-FISH showed the subcellular localization of RHPN1-AS1 in ES-2 and HEY cells. RHPN1-AS1 was stained with Cy3 (red) and nuclei were stained with DAPI (blue). (C) Ago2-RIP was performed in ES-2 and HEY cells. (D, E) Enrichment of RHPN1-AS1 in ES-2 and HEY cells transfected with miR-596 mimic. (F) Prediction of miR-596 binding site in the RHPN1-AS1. (G) Luciferase activity in ES-2 and HEY cells co-transfected with WT/mut RHPN1-AS1 plasmids, and miR-596 mimic or miR-Ctrl. (H) Biotin-miR-596 pull-down enrichment of RHPN1-AS1. (I) Relative levels of miR-596 in ES-2 and HEY cells transfected with sh-RHPN1-AS1#2 or scrambled control. (J) Relative levels of RHPN1-AS1 in ES-2 and HEY cells transfected with miR-596-mimic or miR-Ctrl. *P<0.05, **P<0.01.