Effects of miR-204-5p modulation on PAX6 regulation and corneal inflammation

Abstract

Congenital aniridia is a rare eye disease characterized by loss of PAX6 protein leading to aniridia-associated keratopathy that significantly reduces vision. The miR-204-5p is a possible regulator of PAX6 function and here we evaluate its effect in multiple in vitro and in vivo models. In vitro, miR-204-5p overexpression suppressed vascular factor ANGPT1 in human limbal stem cells (T-LSC) and Pax6-knockdown LSC (mut-LSC), and in primary human limbal epithelial cells (LEC) at the gene and protein levels and following LPS stimulation. However, miR-204-5p inhibited VEGFA expression only in mut-LSCs and LPS-stimulated LEC. Also, miR-204-5p increased PAX6 expression in mut-LSC and differentiated corneal epithelial cells, but not in LEC. Topical miR-204-5p after LPS-induced keratitis in mice failed to suppress Vegfa, Angpt1, Il-1β, and Tnf-α or rescue Pax6 levels in contrast to in vitro results, although it significantly reduced the inflammatory infiltrate in the cornea. In Pax6^Sey/+ aniridia mice, miR-204-5p did not rescue PAX6 levels or suppress Vegfa, Angpt1, or inhibit the ERK1/2 pathway. While short-term miR-204-5p treatment effectively suppresses VEGFA and ANGPT1 and enhances PAX6 expression in multiple corneal epithelia, effects are variable across primary and immortalized cells. Effects of longer-term in vivo treatment, however, require further study.

Keywords: Aniridia; Corneal inflammation; Keratitis; MiR-204-5p; PAX6.

Fig. 1

Comparative analysis of miR-204-5p expression levels in human corneal epithelial cell lines and mouse corneas on different backgrounds. qRT-PCR analysis revealed significantly lower miR-204-5p expression in T-LSC, mut-LSC, and HCE-T relative to the corneas of C57BL/6 (p < 0.0001) and 129S1/SvlmJ strains (p = 0.005 for T-LSC and mut-LSC and p = 0.009 for HCE-T). Corneas of the WT-129S mice showed higher levels of miR-204-5p relative to the Pax6^+/− corneas of littermates (p = 0.02).

Fig. 2

miR-204-5p administration in T-LSC and mut-LSC modulates levels of PAX6 and canonical vascular growth factors. (A) Live/dead cytotoxic assay showed no cytotoxicity of miR-204-5p in T-LSC and mut-LSC cells. Data are presented as mean ± SD of the percentage of survival. (B) miR-204-5p was significantly increased in both T-LSC (p < 0.001) and mut-LSC (p < 0.01) 24 h after transfection. (C) PAX6 expression was significantly elevated in mut-LSC following miR-204-5p transfection (p = 0.007) but not in T-LSC (p = 0.02). (D,E) Increased levels of miR-204-5p stimulated downregulation of VEGFA (p = 0.003) and ANGPT1 (p = 0.04) in mut-LSC, while ANGPT1 expression also declined in T-LSC (p = 0.01). miR-204 denotes miR-204-5p in the graph labels.

Fig. 3

miR-204-5p modifications resulted in PAX6 higher levels in HCE-T cells. (A) There was a statistically significant level of miR-204-5p 24 h post-transfection in this non-limbal corneal epithelial cell line (p = 0.03). (B) qRT-PCR verification indicated an increased level of PAX6 following the cells being treated with miR-204-5p (p = 0.02). miR-204 denotes miR-204-5p in the graph labels.

Fig. 4

miR-204-5p modulation of gene and expression in primary LECs. (A) miR-204-5p level significantly elevated following the transfection (p < 0.001). (B–D) While mRNA expression levels of PAX6 and VEGFA did not show remarkable alterations (p = 0.08, 0.16, respectively), ANGPT1 was significantly downregulated (p = 0.03) following transfection with miR-204-5p in primary LEC. (E–G) A similar trend was observed in protein levels via western blot of PAX6 (p = 0.14), VEGFA (p = 0.13), and ANGPT1 (p = 0.03) in primary LEC following miR-204-5p transfection. miR-204 denotes miR-204-5p in the graph labels.

Fig. 5

Effect of miR-204-5p treatment in LPS-stimulated primary LEC. (A) miR-204-5p showed significantly higher levels following transfection (p < 0.0001). (B–D) mRNA expression levels of VEGFA and ANGPT1 indicated a significant decline after being treated with miRNA in the LPS condition (p = 0.006 and p = 0.004, respectively) while the PAX6 level remained constant. (E–G) Protein expression analysis revealed a significant decrease in ANGPT1 (p = 0.01) after miRNA treatment in the LPS condition while VEGFA (p = 0.052) and PAX6 (p = 0.48) did not present significant alterations. miR-204 denotes miR-204-5p in the graph labels.

Fig. 6

miR-204-5p treatment contributed to partial in vivo amelioration of LPS-mediated corneal keratitis. (A) subconjunctival co-injection of miRNA and LPS. (B) 48 h after LPS induction and after QID miR-204-5p topical treatment, corneal tissues had a significantly elevated level of miR-204-5p relative to the non-treated control group (p = 0.03). (C) Representative slit-lamp images from different clinical opacification grades were observed among both treated groups. (D) No significant alteration in corneal transparency was noted among experimental groups 48 h post-induction (E, F) LPS injection stimulated an inflammatory infiltrate within corneal layers and miR-204-5p treatment significantly reduced the cell infiltration in the epithelium and anterior stroma (p < 0.0001, p < 0.01 respectively).

Fig. 7

Effect of short duration miR-204-5p treatment in an acute model of LPS-induced keratitis. (A) A decline in Pax6 mRNA level (p < 0.0001) following LPS-induced keratitis in mice cornea was observed, but miR-204-5p treatment for 48 h failed to mitigate this decline. (B,C) Following LPS stimulation, Vegfa mRNA increased (p = 0.04) while a non-significant increasing trend in Angpt1 levels was also noted, with neither being mitigated by miR-204-5p treatment. (D,E) Despite observing significant upregulation in inflammatory factors Il-1β (p = 0.003) and Tnf-α (p = 0.008) following LPS-induced keratitis, miR-204-5p treatment for 48 h did not suppress these levels. (miR-204 denotes miR-204-5p in the graph label).

Fig. 8

Effects of short-duration miR-204-5p treatment at the mRNA and protein level in the Pax6^Sey/+ aniridia mouse model. (A) miR-204-5p levels were considerably downregulated in the corneas of the Pax6^Sey/+ aniridia mice relative to WT-129S mice with similar backgrounds (p < 0.001) but this was not altered by short-duration miR-204-5p treatment. (B) miR-204-5p treatment correspondingly did not change Pax6 mRNA expression (p = 0.8). (C,D) In Pax6^Sey/+ mice, Vegfa and Angpt1 markers were significantly upregulated compared to WT-129S controls while miRNA treatment similarly did not change these levels (p = 0.02 and p = 0.01, respectively). (E) Following miR-204-5p treatment in the model, pERK1/2 protein was not suppressed (p = 0.4). (F) Accordingly, the PAX6 protein level was unchanged (p = 0.1). miR-204 denotes miR-204-5p in the graph labels.