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.
Fig 2: Age- dependent correlation of HOXA5, PTEN and p53 mRNA expression. (A) mRNA expression levels of HOXA5, PTEN and p53 are downregulated in the old subjects compared to the young cohort. (B) Moderate to strong linear correlations were observed between HOXA5 and PTEN mRNA expression (r = 0.7115) (C) between HOXA5 and p53 mRNA expression (r = 0.6294), and (D) between p53 with PTEN mRNA expression (r = 0.6685). Detection of mRNA expression was performed by qRT-PCR on total cellular RNA prepared from monocytes. Error bars denote SEM (n = 30, average age: 23.0 yrs and 81.1 yrs, respectively).
Fig 3: Clinical relevance of cZYG11B-mediated signaling in I/R-related ocular disease. Aqueous humor samples were collected from patients with glaucoma (n=20) and patients with cataracts (Ctrl; n=20). Reverse transcription-quantitative PCR was conducted to detect the expression levels of (A) cZYG11B and (B) miR-620. (C) ELISA was conducted to detect the concentration of PTEN. The significant differences were determined by unpaired Student's t-test. *P<0.05 vs. Ctrl group. Ctrl, control; cZYG11B, circZYG11B; miR, microRNA.
Fig 4: Glyoxalase 1 (Glo1) upregulation is driven by the PI3/AKT/mTOR pathway. (a,c) Ectopic expression of PTEN (pCMV-PTEN) in PC3 cells or (b,d) PTEN knockdown (siPTEN) in DU145 cells led to desensitization or activation of the PI3K/AKT/mTOR pathway, respectively. Treatment of PC3 cells with the PI3K inhibitor LY294002 (LY), the AKT inhibitor MK2206 (MK), and the mTOR inhibitor rapamycin (Rapam) further confirmed the involvement of the PI3K/AKT/mTOR signaling cascade in regulating (e) Glo1 at both the transcript and the functional level. The PI3/AKT/mTOR pathway was evaluated by p-AKT and p-mTOR expression measured by both specific ELISA kits and Western blotting. mRNA expression was evaluated by qRT-PCR, while specific enzyme activity was measured by a specific spectrophotometric method. β-actin served as the internal control. The histograms indicate the mean ± SD of three different cultures. pCMV-Ctr: control containing plasmid DNA; siCtr: control (non-specific siRNA). ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Fig 5: The desensitization of the MG-H1/RAGE/H2O2/KRIT1 axis sustains the growth of aggressive PC3 cells. Glo1 overexpression, driven by the PTEN/PKM2/ERα pathway, reduces intracellular MG-H1 levels, which desensitizes RAGE signaling and consequently decreases H2O2 production. This reduction leads to KRIT1 downregulation, thereby promoting cellular growth. Created with www.BioRender.com.
Supplier Page from Abcam for Human PTEN ELISA Kit