Modulation of inflammatory markers by miR-146a during replicative senescence in trabecular meshwork cells.
OBJECTIVE
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.
Li
Guorong
G
Department of Ophthalmology, Duke University, Durham, North Carolina 27710, USA.
Luna
Coralia
C
Qiu
Jianming
J
Epstein
David L
DL
Gonzalez
Pedro
P
D000328:Adult; D015153:Blotting, Western; D002460:Cell Line; D049109:Cell Proliferation; D016922:Cellular Senescence / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D005819:Genetic Markers; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial; D035683:MicroRNAs / Q000235:genetics*; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014019:Tissue Donors; D014129:Trabecular Meshwork / Q000166:cytology* / Q000378:metabolism*; D014162:Transfection
D005819:Genetic Markers; D015850:Interleukin-6; D016209:Interleukin-8; C542542:MIRN146 microRNA, human; D035683:MicroRNAs; D017395:Plasminogen Activator Inhibitor 1; D017382:Reactive Oxygen Species; C051321:SERPINE1 protein, human; C507768:IRAK1 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases