Role of miR-29b on the regulation of the extracellular matrix in human trabecular meshwork cells under chronic oxidative stress.
PURPOSE: To investigate the role of miR-29b on the changes in expression of genes involved in the synthesis and deposition of extracellular matrix (ECM) induced by chronic oxidative stress in human trabecular meshwork cells (HTM). METHODS: Changes in gene expression induced by miR-29b in HTM cells were evaluated by gene array analysis using Affymetrix U133A2 arrays and confirmed by quantitative-PCR. Pathway analysis was conducted using Metacore. Targeting of miR-29b to the 3'-untranslated region of three novel putative targets was evaluated using the Psicheck luciferase system. Chronic oxidative stress was induced by incubation at 40% oxygen for 4-5 days, using cultures incubated at 5% oxygen as controls. Changes in expression in microRNA or gene expression were analyzed by Q-PCR. Cell viability was measured by lactate dehydrogenase release. RESULTS: Transfection of HTM cells with miR-29b mimic resulted in downregulation of multiple ECM components, including collagens (COL1A1, COL1A2, COL4A1, COL5A1, COL5A2, COL3A1) LAMC1, and FBN as well as several genes involved in ECM deposition and remodeling, such as SPARC/osteonectin. Three additional genes, BMP1, ADAM12, and NKIRAS2, were identified as direct targets of miR-29b. Chronic oxidative stress induced a significant downregulation of miR-29b in two HTM cell lines that was associated with increased expression of several ECM genes known to be regulated by miR-29b. The increase in expression of these genes was inhibited by transfection with miR-29b mimic. MiR-29b increased cell viability under both chronic oxidative stress and physiologic oxygen concentrations. CONCLUSIONS: MiR-29b negatively regulates the expression of multiple genes involved in the synthesis and deposition of ECM in trabecular meshwork (TM) cells. Downregulation of miR-29b might contribute to increased expression of several ECM genes under chronic oxidative stress conditions. The balance between the activation of ECM production induced by oxidative stress and the protective effects of miR-29b could be a relevant factor in understanding how oxidative damage may lead to increased deposition of ECM in the TM and contribute to the elevation of intra-ocular pressure in glaucoma.
Luna, C; Li, G; Qiu, J; Epstein, DL; Gonzalez, P
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