Effect of isolated keratin 3 knockdown on gene expression of primary limbal epithelial cells without and with inflammatory stimuli.

Objective: Studies have shown that keratin 3 (KRT3) expression is reduced in paired box 6 (PAX6) haploinsufficient primary limbal epithelial cells (LECs). The downregulation of KRT3 expression due to PAX6 haploinsufficiency is likely a critical factor in the development and progression of aniridia associated keratopathy (AAK). In addition, the ocular surface of congenital aniridia patients exhibits an inflammatory environment. The objective of this study was to investigate the isolated effect of KRT3 knockdown, achieved via siRNA silencing in healthy LECs, on PAX6 and other related gene expressions, both under normal and inflammatory conditions.

Methods: To achieve KRT3 knockdown, human primary LECs were transfected with KRT3 siRNA using Lipofectamine 2000. Inflammatory conditions were induced 48hours after transfection by treating the cells with 2mg/mL of lipopolysaccharides (LPS) or 1ng/mL of IL-1β. Subsequently, gene and protein expression levels were analysed using qPCR, Western blotting, and ELISA.

Results: Following KRT3 knockdown at protein level, there was DSG1, ADH7 and PPARγ upregulation and MAPK1 downregulation solely at transcriptional level (p≤0.031). Nevertheless, IL-6 downregulation could be observed both at transcriptional and at protein levels (p≤0.003). Following KRT3 siRNA knockdown, LPS induced inflammation decreased PPARγ mRNA level and IL-1β induced inflammation decreased DSG1 and ADH7 mRNA levels without changes at protein levels (p≤0.014). In contrast, in control knockdown LECs, IL-1β induced inflammation significantly decreased KRT3 mRNA and protein levels and IL-6 protein level (p≤0.02).

Conclusions: In normal LECs, inflammatory stimuli slow differentiation and simultaneously induce IL-6 production. These mechanisms are absent in KRT3 knockdown LECs. As a result, despite the presence of inflammation, KRT3 knockdown LECs continue their differentiation unaltered while maintaining inflammatory IL-6 protein secretion.