LEF1 influences diabetic retinopathy and retinal pigment epithelial cell ferroptosis via the miR-495-3p/GRP78 axis through lnc-MGC

Figure 1 Differential expression of lnc-MGC and miR-495-3p in the diabetic retinopathy rat retina and cell model. A: Detection of lnc-MGC and miR-495-3p expression in diabetic retinopathy rat retina tissue by RT-qPCR; B: Expression levels of lnc-MGC and miR-495-3p in the cell model were detected by RT-qPCR; C: Localization of lnc-MGC in ARPE-19 cells was detected by fluorescence in situ hybridization (FISH). The scale bar represents 10 μm; D: Localization of miR-495-3p in ARPE-19 cells was detected by FISH. The scale bar represents 10 μm. ^aP < 0.001. NC: Normal control; DR: Diabetic retinopathy; HG: High glucose.

Figure 2 Inhibition of lnc-MGC protects retinal pigment epithelial cells. A: RT-qPCR was used to detect the transfection efficiency; B: CCK-8 was used to detect cell proliferation activity; C: TUNEL was used to detect cell apoptosis. The scale bar represents 100 μm; D: ELISA was used to detect the expression of the inflammatory factors TNF-α, IL-6 and IL-1β; E: Flow cytometry was used to detect reactive oxygen species; F: Western blotting was used to detect the expression of the apoptosis-related proteins Bax, Bcl-2 and cleaved-caspase 3. ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001. NC: Normal control; HG: High glucose; ROS: Reactive oxygen species.

Figure 3 Downregulation of lnc-MGC can mediate the protective effect of retinal pigment epithelial cells through ER stress and ferroptosis. A: CCK-8 detection of cell proliferation activity; B: TUNEL detection of cell apoptosis. The scale bar represents 100 μm; C: ELISA detection of the expression of the inflammatory cytokines TNF-α, IL-1β and IL-6; D: Flow cytometry detection of reactive oxygen species; E: Western blotting was used to detect the expression of the apoptosis-related proteins Bax, Bcl-2 and cleaved-caspase 3; F: Western blotting was used to detect the expression of the ER-related proteins GRP78, CHOP and XBP-1; G: Western blotting was used to detect the expression of the ferroptosis-associated proteins GPX4, HO-1, FTH1, and xCT. ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001. NC: Normal control; HG: High glucose; ROS: Reactive oxygen species.

Figure 4 Correlation between lnc-MGC and miR-495-3p. A: The StarBase was used to predict the binding sites of lnc-MGC and miR-495-3p; B: Dual-luciferase gene analysis was used to verify the targeting binding relationship between lnc-MGC and miR-495-3p; C: The expression of miR-495-3p was detected by RT-qPCR. ^aP < 0.001, ^bP < 0.01. WT: Wild type; MUT: Mutation; NC: Normal control; HG: High glucose.

Figure 5 lnc-MGC affects the progression of diabetic retinopathy through miR-495-3p. A: Transfection efficiency was detected by RT-qPCR; B: Cell proliferation was detected by CCK-8 assay; C: Cell apoptosis was detected by TUNEL assay. The scale bar represents 100 μm; D: The expression of the inflammatory cytokines TNF-α, IL-1β, IL-6 was detected by ELISA; E: reactive oxygen species were detected by flow cytometry; F-H: Western blotting was used to detect the expression of related proteins. ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001. Inhibitor: MiR-495-3p inhibitor; NC: Normal control; HG: High glucose; ROS: Reactive oxygen species.

Figure 6 miR-495-3p targets GRP78. A: The TargetScan was used to predict binding sites for miR-495-3p and GRP78; B: Dual-luciferase verification that miR-495-3p targets GRP78. ^aP < 0.01. WT: Wild type; MUT: Mutation; NC mimic: Negative control mimic.

Figure 7 miR-495-3p regulates endoplasmic reticulum stress and ferroptosis through GRP78. A: RT-qPCR was used to detect transfection efficiency; B: Western blot was used to detect transfection efficiency; C: CCK-8 was used to detect cell proliferation activity; D: A TUNEL assay was used to detect apoptosis. The scale bar represents 100 μm; E: The expression of TNF-α, IL-1β and IL-6 was detected by ELISA; F: Reactive oxygen species were detected by flow cytometry; G-I: Western blotting was used to detect the expression of related proteins. ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001. NC: Normal control; HG: High glucose; ROS: Reactive oxygen species; mimic: miR-495-3p mimic.

Figure 8 LEF1, as the binding protein of lnc-MGC, regulates the progression of diabetic retinopathy. A and B: LEF1 can bind to the lnc-MGC promoter; C: Western blotting was used to detect the inhibition efficiency of LEF1; D: RT-qPCR was used to detect the transfection efficiency; E: CCK-8 was used to measure the cell proliferation activity; F: TUNEL was used to detect cell apoptosis. The scale bar represents 100 μm; G: ELISA was used to determine the expression of the inflammatory cytokines TNF-α, IL-1β and IL-6; H: Flow cytometry was used to detect reactive oxygen species; I-K: Western blotting was used to detect the expression of related proteins. ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001. NC: Normal control; HG: High glucose; ROS: Reactive oxygen species.

Figure 9 Animal experiments to verify that LEF1 combined with lnc-MGC regulates diabetic retinopathy progression. A and B: Diabetic retinopathy rat body weight and blood glucose detection; C: A TUNEL assay was used to detect apoptosis. The scale bar represents 100 μm; D: ELISA was used to detect the expression of the inflammatory factors TNF-α, IL-1β and IL-6; E: Reactive oxygen species were detected by flow cytometry; F-H: Western blotting was used to detect the expression of related proteins; I: HE staining was used to observe the retinal structure. The scale bar represents 20 μm. ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001. ONL: Outer nuclear layer; INL: Inner core layer; RGC: retinal ganglion cells; NC: Normal control; DR: Diabetic retinopathy; ROS: Reactive oxygen species.