Transcriptomic and proteomic analyses of sclera in lens-induced myopic guinea pigs

Abstract

Background: Myopia development is commonly assessed by an increase in axial length, which may lead to high myopia and visual impairment. This study aims to identify potential biomarkers and signaling pathways in the sclera during experimental axial elongation.

Methods: A myopia guinea pig model was established using male guinea pigs aged 2-3 weeks, which underwent bilateral lens-induced myopization (LIM) (study group) or were left untreated (control group). An integrated analysis of transcriptomic and proteomic was performed to identify differentially expressed genes (DEGs) in the sclera. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted to explore the DEGs related signaling pathways. Promising candidate markers were further tested by Western blot analysis. Transmission electron microscopy was used to assess scleral fiber changes in myopic guinea pigs.

Results: During the study period, axial elongation was significantly greater in the study group (0.59 ± 0.05 mm vs. 0.47 ± 0.02 mm; P < 0.001), accompanied by a reduction in the thickness of the retina (121.9 ± 2.50 μm vs. 134.6 ± 0.48 μm; P < 0.001), choroid (38 ± 1.0 μm vs. 50 ± 0.8 μm; P < 0.001), and sclera (100.8 ± 2.78 μm vs. 155.6 ± 4.78 μm; P < 0.001). Integrated transcriptomic and proteomic analyses identified 34 upregulated genes, with significant activation and enrichment of the circadian rhythm pathway. Among the top enriched pathways, key differentially expressed genes included retinoid-related orphan receptors RORα and RORβ, which are recognized as critical signals modulating the scleral hypoxia response. Western blot analysis confirmed upregulation of RORα, RORβ, melatonin receptor type 1 (MT1), and HIF-1α in the sclera, while melatonin receptor type 2 (MT2) expression remained unchanged between the groups. Transmission electron microscopy revealed a significantly higher proportion of thin collagen fibers compared to thick fibers in the LIM group (P < 0.05).

Conclusions: Axial elongation-related remodeling of scleral collagen is closely linked to circadian rhythm and hypoxia pathways, with RORα, RORβ, melatonin receptors, and HIF-1α identified as potential key regulators. Additionally, scleral fiber size decreases progressively with scleral remodeling in myopia development.

Keywords: Lens-Induced myopia; Melatonin receptors; Proteomics; Retinoid-related orphan receptors (RORs); Transcriptomics.

Fig. 1

A: Changes in axial length from study baseline to study end between the lens-induced myopia (LIM) study group and control group. B: Thickness of the posterior retina measured at 1000 μm temporal to the optic disc at study end. C: Thickness of the posterior choroid measured at 1000 μm temporal to the optic disc at study end. D: Thickness of the posterior sclera measured at 1000 μm temporal to the optic disc at study end. E, F: Optical coherence tomographic images of the posterior pole of guinea pigs at the study end, in the control group (E) and the LIM study group (F). scale bar: 100 μm. ***: P < 0.001

Fig. 2

A: Volcano plot showing differently expressed genes in guinea pigs of the lens-induced myopia (LIM) study group and control group. B, C: Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses identifying the most significant affected pathways in the LIM study group (P < 0.05)

Fig. 3

A: Volcano plot shows differently expressed proteins in guinea pigs of the lens-induced myopia (LIM) study group and control group. B, C: Gene Ontology (GO) of chordal graph and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses enrichment analysis identifying the most significant affected pathways in the LIM study group, indicating the overexpression of genes involved in RORα-related and RORβ-related circadian rhythms pathways (P < 0.05)

Fig. 4

Comparison of the changes in mRNA and protein in guinea pigs of the lens-induced myopia (LIM) study group and control group. A: A total of 34 genes were upregulated in both the transcriptomic and proteomic analyses, 16 genes downregulated in both comparisons, 32 downregulated mRNA that were upregulated at the protein level, and 45 gene upregulated at the mRNA level but downregulated at the proteins level with a correlation of 0.03 (green) (P < 0.05). We identified 79 significantly upregulated and 79 downregulated proteins with no mRNA expression changes (pink) (P < 0.05). A total of 229 significantly upregulated and 315 downregulated mRNA genes with no proteins expression changes (purple) (P < 0.05). B: The top 10 most significant proteins as revealed by String analysis mainly participated in inflammation activities. LIM: lens-induced myopia

Fig. 5

Circadian rhythms pathway in the sclera of guinea pigs of the lens-induced myopia (LIM) study group and control group. A-E: Expression level of RORα, RORβ, melatonin receptors type1 (MT₁), melatonin receptors tyep2 (MT₂) and HIF-1α in the sclera. F, G: Transmission electron microscopical images of sclera collagen fibers in the control group (F) and the LIM (G) study group; scale bar, 5 μm. H: Percentage of sclera fiber in different diameters (n = 3 biologically independent samples). Five images from one sclera. LIM: lens-induced myopia; MT₁: melatonin receptors type1; MT₂: melatonin receptors type2. *: P < 0.05; **: P < 0.01; ns: not statistically significant