Purpose

MiR-146a upregulated in limbus vs. central cornea and in diabetic vs. non-diabetic limbus has emerged as an important immune and inflammatory signaling mediator in corneal epithelial wound healing. Our aim was to investigate the potential inflammation-related miR-146a target genes and their roles in normal and impaired diabetic corneal epithelial wound healing.

Methods

Our previous data from RNA-seq combined with quantitative proteomics of limbal epithelial cells (LECs) transfected with miR-146a mimic vs. mimic control were analyzed. Western blot and immunostaining were used to confirm the expression of miR-146a inflammatory target proteins in LECs and organ-cultured corneas. Luminex assay was performed on conditioned media at 6- and 20-h post-wounding in miR-146a mimic/inhibitor transfected normal and diabetic cultured LECs.

Results

Overexpression of miR-146a decreased the expression of pro-inflammatory TRAF6 and IRAK1 and downstream target NF-κB after challenge with lipopolysaccharide (LPS) or wounding. Additionally, miR-146a overexpression suppressed the production of downstream inflammatory mediators including secreted cytokines IL-1α, IL-1β, IL-6 and IL-8, and chemokines CXCL1, CXCL2 and CXCL5. These cytokines and chemokines were upregulated in normal but not in diabetic LEC during wounding. Furthermore, we achieved normalized levels of altered secreted cytokines and chemokines in diabetic wounded LEC via specific inhibition of miR-146a.

Conclusion

Our study documented significant impact of miR-146a on the expression of inflammatory mediators at the mRNA and protein levels during acute inflammatory responses and wound healing, providing insights into the regulatory role of miR-146a in corneal epithelial homeostasis in normal and diabetic conditions.

中文翻译：

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miR-146a 通过其在人类糖尿病角膜中的炎症靶点在角膜上皮伤口愈合中的调节作用

目的

MiR-146a 在角膜缘与中央角膜以及糖尿病与非糖尿病角膜缘中上调已成为角膜上皮伤口愈合中重要的免疫和炎症信号传导介质。我们的目的是研究潜在的炎症相关 miR-146a 靶基因及其在正常和受损糖尿病角膜上皮伤口愈合中的作用。

方法

我们之前的 RNA-seq 数据与转染 miR-146a 模拟物与模拟物对照的角膜缘上皮细胞 (LEC) 的定量蛋白质组学相结合进行了分析。蛋白质印迹和免疫染色用于确认 miR-146a 炎症靶蛋白在 LEC 和器官培养的角膜中的表达。在 miR-146a 模拟物/抑制剂转染的正常和糖尿病培养的 LEC 中受伤后 6 小时和 20 小时，在条件培养基上进行 Luminex 测定。

结果

在脂多糖(LPS)攻击或受伤后，miR-146a 的过表达降低了促炎性 TRAF6 和 IRAK1 以及下游靶标 NF-κB的表达。此外，miR-146a 过表达抑制了下游炎症介质的产生，包括分泌性细胞因子 IL-1α、IL-1β、IL-6 和 IL-8，以及趋化因子 CXCL1、CXCL2 和 CXCL5。这些细胞因子和趋化因子在正常人中上调，但在糖尿病 LEC 中没有上调。此外，我们通过特异性抑制 miR-146a，使糖尿病损伤的 LEC 中分泌的细胞因子和趋化因子水平正常化。

结论

我们的研究记录了 miR-146a 在急性炎症反应和伤口愈合过程中对 mRNA 和蛋白质水平的炎症介质表达的显着影响，提供了对 miR-146a 在正常和糖尿病条件下角膜上皮稳态中的调节作用的见解。