lncRNA ZFAS1 Positively Facilitates Endothelial Ferroptosis via miR‐7‐5p/ACSL4 Axis in Diabetic Retinopathy

Figures

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  Figure 1. ZFAS1 is upregulated in hRECs under HG and in FVM tissues. (a) The volcano map of differentially expressed lncRNAs in GEO: GSE94019. (b) The expression level of lncRNA ZFAS1 in endothelial cells isolated from FVM samples and healthy control retinal samples. (c) RT-PCR was performed to detect the ZFAS1 expression cultured in LG (5.5 mM), HG (10, 15, 20, and 30 mM), or hypertonic control group (5.5 mM glucose and 24.5 mM mannitol) medium for 48 h. n = 5 replicates. Data are presented as ± SEM. ∗∗∗P < 0.001.

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  Figure 2. Inhibition of ZFAS1 rescued HG-induced ferroptosis in hRECs. (a) Both apoptosis inhibitor TUDCA and ferroptosis inhibitor Fer-1 administration rescued the downregulated cell viability in HG group. Compared to HG control, there is no difference in total (b) or ferrous iron level (c), lipid hydroperoxide accumulation (d and e), nor GPx4 expression level (f) after 10 μM apoptosis inhibitor TUDCA, necrosis inhibitor necrostatin-1, pyroptosis inhibitor TETD treatment for 48 h, while transfection of Sh-ZFAS1 remarkably lightened the above ferroptosis phenotypes. n = 5 replicates. P > 0.05. 10 μM Fer-1 treatment for 48 h notably ameliorated the HG-induced ferroptosis-related phenotypes indicated above. n = 5 replicates. Data are presented as ± SEM. Ns: no significant; ∗∗P < 0.01 and ∗∗∗P < 0.001. Scale bar, 50 μm.

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  Figure 3. ZFAS1 regulates ACSL4 expression by directly interacting with miR-7-5p. (a) The binding sites of ZFAS1 with miR-7-5p as predicted by starBase. (b) Transfection of miR-7-5p mimics remarkably attenuated the luciferase activity of ZFAS1-WT compared with negative control groups. (c) RT-qPCR was employed to detect the expression of lncRNA ZFAS1 in the cytoplasm and nucleus of hRECs. GAPDH and U6 served as cytoplasmic and nuclear markers, respectively. n = 5 in each group. (d) Colocalization between ZFAS1 (labeled in red) and miR-7-5p (labeled in green) was observed by RNA FISH in hRECs. Scale bar, 25 μm. (e) hRECs were transfected with empty vector pcDNA3.1 or pcDNA3.1-ZFAS1 for 48 h, and the expression level of miR-7-5p was validated using RT-qPCR. miR-39-3p served as an exogenous normalization. n = 5 in each group. (f) Five different datasets were utilized to predict the downstream molecule of miR-7-5p. (g) The binding sites of miR-7-5p with ACSL4 as predicted by starBase. All data are presented as ± SEM. ∗∗∗P < 0.001. (h) miR-7-5p mimics remarkably attenuated the luciferase activity of ACSL4-WT compared with negative control groups.

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  Figure 4. The effect of ZFAS1 silencing on preventing ferroptosis was abolished by dual knockdown of ZFAS1 and miR-7-5p. RT-qPCR results demonstrated the miR-7-5p (a) and ACSL4 expression level (b). GAPDH and miR-39-3p served as endogenous and exogenous normalization for ACSL4 and miR-7-5p detection, respectively. n = 5 replicates. Data are presented as ± SEM. ∗∗∗P < 0.001. (c–g) hRECs were transfected with 50 μM Sh-ZFAS1 or together with 50 μM miR-7-5p inhibition, and the cell viability (c), total (d), ferrous iron level (e), and lipid hydroperoxide accumulation (f and g) were assessed 72 hours after the transfection. n = 5 replicates. Data are presented as ± SEM. ∗∗∗P < 0.001. Scale bar, 50 μm. (h) Analysis of GPx4 and ACSL4 expression level.

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  Figure 5. miR-7-5p knockdown partially reverses the functional effect of ZFAS1 knockdown on endothelial ferroptosis in DR mice. (a) Schematic representation of STZ administration. C57BL/6J male mice at 6 weeks old were randomly separated into four experimental groups. Streptozotocin was injected intraperitoneally at a final concentration of 55 mg/kg for 5 consecutive days to induce diabetes, and all diabetic mice were fed with high-fat diet subsequently. Six weeks after the final injection, indicated plasmids (including 100 nM Sh-ZFAS1 and 50 nM Inhi-miR-7-5p) or vehicles were intravitreally injected once a week for four weeks. RT-qPCR validated the expression level of ZFAS1 (b), miR-7-5p (c), and ACSL4 (d). (e) Western blotting analysis validated the ACSL4 protein level. (f) Immunofluorescence assay for GPx4 (labelled red) expression in mouse retinal endothelial cell (labelled green). Scale bar for retinal flat mounts, 50 μm. Scale bar for retinal cross section, 50 μm. (g) Quantification of GPx4 intensity. Data are presented as ± SEM. ∗∗∗P < 0.001. (h) The summary diagram. ZFAS1 was induced under HG condition. It functioned as a “ceRNA” by sponging miR-7-5p, thus upregulating ACSL4…