We initially performed a toxicology study involving siHOTAIR. Wild-type C57BL/6J mice were subjected to a one-time intravitreal injection of either SCR siRNA (100 nM) or siHOTAIR at varying concentrations (25, 50, or 100 nM) and were monitored for 7 days when tissues were collected. After 7 days, retinal
HOTAIR expression appeared to be the lowest following a 100-nM dose of siHOTAIR (∼50% reduction) when compared to SCR controls (
Supplementary Fig. S8A). No behavioral changes were observed, and analysis of H&E stained sections showed no structural abnormalities across retinal, heart, lung, liver, or kidney tissues (
Supplementary Figs. S8B–F). Hence, for therapeutic assessment, 100 nM of siHOTAIR or SCR was intravitreally injected weekly in diabetic mice showing hyperglycemia and loss of body weight (
Supplementary Figs. S9A, B), and
HOTAIR knockdown did not affect these parameters. However, at 4 weeks, elevated RNA expression of
HOTAIR,
Vegf-a,
Ctcf,
Et-1,
Angptl4,
Mcp-1,
Il-1β,
Pgf,
Hif-1α,
p300, polycomb repressive complex 2 (
Prc2) components (
Ezh2,
Suz12, and
Eed),
Hoxd3, and
Parp1 were seen in the retina of diabetic mice administered SCR siRNAs (
Fig. 4;
Supplementary Figs. S9C–I). Knockdown of
HOTAIR (∼58% reduction) reduced diabetes-induced upregulations of most of these transcripts, suggesting that
HOTAIR knockdown protects against early diabetes-induced molecular aberrations in the retina. However, we did not observe differences in retinal expression of
Il-1β,
Pgf, and
Hoxd3 between SCR and siHOTAIR-treated animals (
Figs. 4G;
Supplementary Figs. S9F, I). Retinal vascular permeability was also assessed by immunohistochemical staining for immunoglobulin G (IgG). Our findings demonstrated that IgG was mainly localized within the capillaries, without any significant staining of retinal tissues in non-diabetic animals treated with SCR or
HOTAIR siRNAs (
Fig. 4I, score 1). Conversely, diffuse extravascular IgG staining was observed in the retinal tissues of diabetic animals treated with SCR siRNAs, signifying increased extravasation in the neural retina (
Fig. 4J, score 3), whereas
HOTAIR knockdown protected diabetic animals from such changes (
Fig. 4L, score 1).