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Modulation of inflammatory markers by miR-146a during replicative senescence in trabecular meshwork cells
Guorong Li1, Coralia Luna, Jianming Qiu
1Department of Ophthalmology, Duke University, Durham, North Carolina 27710, USA.
Purpose:
To investigate the alterations in microRNA (miRNA) expression during replicative senescence (RS) in human trabecular meshwork (HTM) cells.
Methods:
Two HTM cell lines were serially passaged until they reached RS. Changes in expression of 30 miRNAs were assessed by real-time quantitative (q)-PCR. The effects of miR-146a on gene expression were analyzed with gene arrays and the results confirmed by real-time q-PCR. Protein levels of IRAK1 and PAI-1 were analyzed by Western blot and those of IL6 and IL8 by ELISA. Senescence-associated markers were monitored by flow cytometry and cell proliferation by BrdU incorporation.
Results:
RS of HTM cells was associated with significant changes in expression of 18 miRNAs, including the upregulation of miR-146a. miR-146a downregulated multiple genes associated with inflammation, including IRAK1, IL6, IL8, and PAI-1, inhibited senescence-associated beta-galactosidase (SA-beta-gal) activity and production of intracellular reactive species (iROS), and increased cell proliferation. Overexpression of either IRAK1 or PAI-1 inhibited the effects of miR-146a on cell proliferation and iROS production in senescent cells.
Conclusions:
RS in HTM cells was associated with changes in miRNA expression that could influence the senescent phenotype. Upregulation of the anti-inflammatory miR-146a may serve to restrain excessive production of inflammatory mediators in senescent cells and limit their deleterious effects on the surrounding tissue. Among the different proteins repressed by miR-146a, the inhibition of PAI-1 may act to minimize the effects of senescence on the generation of iROS and growth arrest and prevent alterations of the extracellular proteolytic activity of the TM.
Insights
Replicative senescence in human trabecular meshwork cells alters microRNA expression. Upregulated miR-146a restrains inflammation and reactive species, potentially protecting ocular tissues.
Area of Science:
- Ophthalmology
- Cell Biology
- Molecular Biology
Background:
- Replicative senescence (RS) is a state of irreversible cell cycle arrest.
- Human trabecular meshwork (HTM) cells play a crucial role in maintaining intraocular pressure.
- Understanding cellular aging in HTM is vital for glaucoma research.
Purpose of the Study:
- To investigate microRNA (miRNA) expression changes during replicative senescence in HTM cells.
- To determine the functional role of specific miRNAs, particularly miR-146a, in the senescent HTM phenotype.
Main Methods:
- Serial passaging of HTM cell lines to induce replicative senescence.
- Real-time quantitative PCR (q-PCR) to assess miRNA expression.
- Gene arrays, Western blot, ELISA, flow cytometry, and BrdU incorporation assays to analyze gene/protein expression and cellular functions.
Main Results:
- RS in HTM cells significantly altered the expression of 18 miRNAs, with miR-146a being upregulated.
- miR-146a downregulated inflammatory genes (IRAK1, IL6, IL8, PAI-1), inhibited senescence markers (SA-beta-gal, iROS), and promoted cell proliferation.
- Overexpression of IRAK1 or PAI-1 counteracted miR-146a's effects on proliferation and iROS.
Conclusions:
- MicroRNA expression changes are associated with the senescent phenotype in HTM cells.
- Upregulated miR-146a acts as an anti-inflammatory factor, mitigating detrimental effects of senescent cells on ocular tissues.
- miR-146a's inhibition of PAI-1 may reduce senescence-induced oxidative stress and preserve extracellular matrix homeostasis in the trabecular meshwork.
