Eye Program, Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
Eye Program, Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA; David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.
Ocul Surf. 2022 Jul;25:92-100. doi: 10.1016/j.jtos.2022.06.001. Epub 2022 Jun 9.
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.
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.
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.
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.
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的 miR-146a 已成为角膜上皮伤口愈合中重要的免疫和炎症信号介质。我们的目的是研究潜在的与炎症相关的 miR-146a 靶基因及其在正常和受损的糖尿病性角膜上皮伤口愈合中的作用。
我们之前对转染 miR-146a 模拟物与模拟物对照的角膜缘上皮细胞（LEC）的 RNA-seq 结合定量蛋白质组学数据进行了分析。Western blot 和免疫染色用于验证 LEC 和器官培养角膜中 miR-146a 炎症靶蛋白的表达。在 miR-146a 模拟物/抑制剂转染的正常和糖尿病培养的 LEC 中，在创伤后 6 小时和 20 小时，对条件培养基进行 Luminex 测定。
miR-146a 的过表达可降低 LPS 或创伤后 TRAF6 和 IRAK1 及其下游靶标 NF-κB 的促炎表达。此外，miR-146a 的过表达可抑制下游炎症介质包括分泌细胞因子 IL-1α、IL-1β、IL-6 和 IL-8 以及趋化因子 CXCL1、CXCL2 和 CXCL5 的产生。这些细胞因子和趋化因子在正常的 LEC 中在创伤时上调，但在糖尿病的 LEC 中则没有。此外，通过特异性抑制 miR-146a，我们使糖尿病创伤的 LEC 中改变的分泌细胞因子和趋化因子水平恢复正常。
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炎症介质的表达在 mRNA 和蛋白质水平上的显著影响，为 miR-146a 在正常和糖尿病条件下角膜上皮稳态中的调节作用提供了见解。
