Fig 1: cZYG11B regulates RGC function by acting as a miR-620 sponge. (A) Total cellular fractions were isolated from RGCs and immunoprecipitated using Ago2 or IgG antibody. The amount of cZYG11B in the IP was determined by RT-qPCR. *P<0.05 as indicated. (B) RGCs were transfected with pGL3-Basic (Ctrl) or Luc-cZYG11B with various miRNA mimics and pRL-TK vector (internal transfection control). Luciferase activity was detected at 24 h post-transfection using the Dual-Luciferase Reporter Assay kit. *P<0.05 vs. Ctrl. (C) Biotinylated miR-620 or miR-95 were transfected into RGCs. After streptavidin capture, the amount of cZYG11B and cZNF236 (negative control) in the input and bound fractions were detected by RT-qPCR. The relative IP/input ratios were plotted. *P<0.05 as indicated. (D) RGCs were transfected with Scr mimic, miR-620 mimic or were untreated. RT-qPCR was conducted to detect PTEN expression. *P<0.05 vs. Ctrl. (E and F) RGCs were left untreated or transfected with Scr mimic, miR-620 mimic, miR-620 mimic + pcDNA 3.1 (Vector) or PTEN-pcDNA 3.1 (PTEN) for 12 h. The medium was replaced and cells were cultured for an additional 12 h at 37°C. These groups were then exposed to 1% O2 to mimic hypoxic stress for 24 h at 37°C. (E) CCK-8 assays were conducted to detect the viability of RGCs. (F) TUNEL staining assays and semi-quantification analysis were conducted to detect the apoptosis of RGCs. Scale bar, 50 µm. *P<0.05 vs. hypoxia group; #P<0.05 as indicated; NS, no significant difference. All significant differences were evaluated by (A and C) Student's t-test or (B, D-F) one-way ANOVA followed by the post hoc Bonferroni test. n=4. Ctrl, control; cZYG11B, circZYG11B; IP, immunoprecipitated; miR, microRNA; RGC, retinal ganglion cell; RT-qPCR, reverse transcription-quantitative PCR; Scr, scrambled.