Abstract
Purpose: :
Similar to other cell types, senescent human trabecular meshwork (HTM) display a senescence associated secretory phenotype (SAPS) that includes an increase in the production of inflammatory markers similar to that observed in the TM of glaucoma donors. Here, we investigated the potential role of microRNAs (miRNAs) miR-155 and miR-146a on the regulation of such chronic activation of inflammatory mediators in senescent HTM cells.
Methods: :
Levels of miRNA expression in replicative senescent HTM cells were evaluated by TaqMan Q-PCR. Changes in gene expression induced by miR-155 and miR146a were analyzed using gene arrays and validated by Q-PCR. Intracellular reactive species (iROS) and senescence associated beta galactosisdase (SA-beta-gal) were measured by flow cytometry, and cell proliferation was determined by BrdU incorporation.
Results: :
Senescent HTM cells showed a decrease in expression of miR-155 (between 4.7±0.07 and -5.3±0.007 fold; p<0.05) and an increase in the expression of miR-146a (between 6.8±0.53 and 111±4.4 fold; p<0.05). MiR-155 down-regulated several potential target genes including CSNK1A1, CSNK1G2 and TCF4, as well as inflammatory mediators such as IL-1beta, ICAM1 and IL7R. Similarly, transfection with miR-146a mimic led to down-regulation of inflammatory markers including IL6, IL8, IL11, CXCL6, CXCL3, CCL20, CCL2, IRAK1 and PAI-1. In addition, miR-146a decreased the expression of the cellular senescence markers SA-beta-gal (-26.4%±3.56; p<0.05) and iROS (-31.1%±1.46; p<0.05), and increased cell proliferation (22.8%±6.82; p<0.05).
Conclusions: :
Changes in expression of both miR-155 and mir-146a, might contribute to regulate the activation of the SASP in senescent HTM cells. While down-regulation of miR-155 in senescent cells might lead to increased production of inflammatory mediators, the up-regulation of miR-146a may be part of a negative feedback loop that restrains excessive production of these inflammatory mediators and limits the potentially deleterious effects of SASP on the surrounding tissue.
Keywords: trabecular meshwork • gene/expression • inflammation