miR-181d-5p Protects against Retinal Ganglion Cell Death after Blunt Ocular Injury by Regulating NFIA-Medicated Astrocyte Development

Abstract

Background: Traumatic optic neuropathy (TON) refers to damage to the optic nerve resulting from direct and indirect trauma to the head and face. One of the important pathological processes in TON is the death of retinal ganglion cells (RGCs), but the cause of RGCs death remains unclear. We aimed to explore the mechanisms of RGCs death in an experimental TON model.

Methods: Optic nerve crush injury was induced in ten New Zealand white rabbits. On the 1st, 3rd, 7th, 14th, and 28th days after the operation, the retinal tissues of the rabbits were observed pathologically by hematoxylin-eosin staining. The expression of POU-homeodomain transcription factor Brn3a and glial fibrillary acidic protein (GFAP) was measured by immunofluorescence to evaluate the number of RGCs and astrocytes, respectively. miRNA expression and protein levels were assessed by RT-qPCR and western blot methods, respectively. Finally, the malondialdehyde content, superoxide dismutase activity, and proinflammatory factor levels were measured by ELISA. Western blot and dual-luciferase reporter assays were used to elucidate the relationship between miR-181d-5p and nuclear factor I-A (NFIA).

Results: Blunt ocular trauma increased oxidative stress and apoptosis and reduced ganglion cell layer (GCL) density. The expression of miR-181d-5p was decreased in retinal tissues, and its overexpression relieved RGCs death, astrocyte development, oxidative stress, and inflammation of the retina, which were reversed by NFIA overexpression.

Conclusion: miR-181d-5p can protect against the deterioration of TON by inhibiting RGCs death, astrocyte development, oxidative stress, and inflammation by targeting NFIA. This study provides new insight into early medical intervention in patients with TON.

Figure 1

Trauma increases retinal oxidative stress and RGCs death. (a) Representative images of retinal tissues stained with hematoxylin and eosin (H&E; scale bar = 40 μm). (b) Superoxide dismutase (SOD) activity. (c) Malondialdehyde (MDA) content. (d) Western blot detection of Bcl-2 and Bax proteins. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 vs. the sham group.

Figure 2

miR-181d-5p is expressed at low levels in injured retinal tissues and RGCs. (a) The RNA-seq data between normal RGCs and injured RGCs (7 days postoptic nerve crush) and selected the top 12 differentially expressed miRNAs. (b) Expression of 12 miRNAs in traumatic retinal tissue by RT-qPCR detection. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 vs. the sham group.

Figure 3

miR-181d-5p relieves RGCs death, astrocyte development, oxidative stress, and inflammation of the retina. (a) Expression of miR-181d-5p by RT-qPCR detection. (b) Representative images of retinal tissues stained by H&E (scale bar = 40 μm). (c) Representative images of POU-homeodomain transcription factor Brn3a staining (scale bar = 40 μm). (d) GFAP staining (scale bar = 40 μm). (e) SOD activity. (f) MDA content. (g) Western blot assay of Bcl-2 and Bax protein. (h) Proinflammatory factor levels were measured by ELISA. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 vs. the sham group; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. the model group.

Figure 4

miR-181d-5p directly targeted the NFIA gene. (a) Representative western blot of nuclear factor I-A (NFIA). ^∗∗P < 0.01 vs. the sham group. (b) The binding sites of miR-181d-5p in the NFIA sequence by starBase prediction. (c) Expression of miR-181d-5p by RT-qPCR detection. ^∗∗∗P < 0.001. (d) The interaction between the NFIA 3′UTR and miR-181d-5p was analyzed by a dual-luciferase reporter gene. 293T cells were cotransfected with miR-181d-5p mimic and WT-NFIA or miR-181d-5p mimic and MUT-NFIA. ^∗∗∗P < 0.001. (e) Representative western blot of NFIA. ^∗∗∗P < 0.001.

Figure 5

miR-181d-5p relieves RGCs death by targeting NFIA. (a) Representative images of retinal tissues stained with H&E (scale bar = 40 μm). (b) Representative images of POU-homeodomain transcription factor Brn3a staining (scale bar = 40 μm). ^∗∗∗P < 0.001; ^#P < 0.05, ^##P < 0.01; ^&P < 0.05, ^&&P < 0.01.

Figure 6

miR-181d-5p relieves astrocyte development, oxidative stress, and inflammation by targeting NFIA. (a) GFAP staining (scale bar = 40 μm). (b) SOD activity. (B) MDA content. (d) Western blot of Bcl-2 and Bax protein. (e) Proinflammatory factor levels were measured by ELISA. ^∗∗P < 0.01 and ^∗∗∗P < 0.001 vs. the sham group; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. the model group; ^&P < 0.05, ^&&P < 0.01, and ^&&&P < 0.001 vs. the miR-181 group.