Modulation of Inflammatory Markers by miR-146a during Replicative Senescence in Trabecular Meshwork Cells

The trabecular meshwork (TM) from glaucoma donors is characterized by chronic activation of a stress response that leads to increased production of inflammatory markers.1–3 Chronic activation of a similar inflammatory response has been found during aging and certain age-related conditions in other tissues.4–12 One of the factors proposed to contribute to such a response is the increased presence of senescent cells. Senescent cells have been shown to accumulate with age and in certain pathologic conditions in several tissues and organs,5,9,13 including the TM in glaucoma.14 The senescent response is associated with a series of phenotypic changes that have been proposed to disrupt the tissue microenvironment and contribute to pathologic alterations associated with aging.15,16 An important alteration observed in senescent cells that may contribute to tissue malfunction is the presence of a characteristic senescence-associated secretory phenotype (SASP).17–19 Such a secretory phenotype involves an increase in the release of inflammatory mediators and growth factors that can affect the function of adjacent cells and lead to a chronic activation of a stress response18 similar to that observed in the TM of glaucoma donors.3 The regulatory mechanisms that mediate the phenotypic changes in senescent cells and, in particular, those involved in the chronic activation of inflammatory mediators have not been completely elucidated. MicroRNAs (miRNAs) are important regulators of gene expression and have been implicated in a variety of cellular functions, including differentiation, apoptosis, and cancer progression.20–22 miRNAs are transcribed as primary transcripts or pri-miRNAs that are converted in the nucleus into 70-nucleotide, stem-loop structures known as pre-miRNAs. These pre-miRNAs are then processed in the cytoplasm to mature miRNAs, 21 to 23 nucleotides in length, by the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC).23 After integration into the active RISC, miRNAs bind to target sites in the 3′ untranslated region (UTR) of the specific mRNA transcripts and inhibit translation or induce mRNA degradation by argonaute proteins, the catalytically active members of the RISC.24 There is some experimental evidence suggesting that miRNAs play a role in cellular senescence. Several miRNAs such as miR-34 and -20a have been shown to induce senescent growth arrest.25,26 Ablation of Dicer in mouse embryonic fibroblasts also induces senescence by upregulating p53.27 Furthermore, we have recently shown that stress-induced premature senescence (SIPS) is associated with significant alteration in expression of several miRNAs in both human fibroblasts and TM cells.28 It has been hypothesized that miRNAs play both positive and negative roles in regulating the senescent response.29–31 Specifically, it has recently been reported that the anti-inflammatory miR-146 is upregulated in senescent fibroblasts in response to increased levels of inflammatory cytokines induced by the senescent response, generating a negative feedback loop that restrains excessive production of inflammatory mediators in senescent cells and limits the deleterious effects of the SASP on the surrounding tissues.31 However, there is still little information about the role that miRNAs play in modulating the senescent response. We investigated changes in expression of miRNAs during the process of replicative senescence (RS) in human TM (HTM) cells. Since one of the miRNAs significantly upregulated during this process was miR-146a, which has been implicated in the modulation of the SASP in fibroblasts,31 we further investigated the effects of this miRNA on gene expression in HTM cells and the mechanisms by which miR-146a may contribute to the senescent response in the TM.