Role of miR-29b on the regulation of the extracellular matrix in human trabecular meshwork cells under chronic oxidative stress

[摘要] Purpose: Toinvestigate the role of miR-29b on the changes in expression of genesinvolved in the synthesis and deposition of extracellular matrix (ECM)induced by chronic oxidative stress in human trabecular meshwork cells(HTM). Methods: Changesin gene expression induced by miR-29b in HTM cells were evaluated bygene array analysis using Affymetrix U133A2 arrays and confirmed byquantitative–PCR. Pathway analysis was conducted using Metacore.Targeting of miR-29b to the 3’-untranslated region of three novelputative targets was evaluated using the Psicheck luciferase system.Chronic oxidative stress was induced by incubation at 40% oxygen for4–5 days, using cultures incubated at 5% oxygen as controls. Changes inexpression in microRNA or gene expression were analyzed by Q-PCR. Cellviability was measured by lactate dehydrogenase release. Results: Transfection of HTM cells withmiR-29b mimic resulted in downregulation of multiple ECM components,including collagens (COL1A1, COL1A2, COL4A1, COL5A1, COL5A2,COL3A1) LAMC1, and FBN as well as several genesinvolved 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 stressinduced a significant downregulation of miR-29b in two HTM cell linesthat was associated with increased expression of several ECM genesknown to be regulated by miR-29b. The increase in expression of thesegenes was inhibited by transfection with miR-29b mimic. MiR-29bincreased cell viability under both chronic oxidative stress andphysiologic oxygen concentrations. Conclusions: MiR-29bnegatively regulates the expression of multiple genes involved in thesynthesis and deposition of ECM in trabecular meshwork (TM) cells.Downregulation of miR-29b might contribute to increased expression ofseveral ECM genes under chronic oxidative stress conditions. Thebalance between the activation of ECM production induced by oxidativestress and the protective effects of miR-29b could be a relevant factorin understanding how oxidative damage may lead to increased depositionof ECM in the TM and contribute to the elevation of intra-ocularpressure in glaucoma.

[学科分类] 生物化学/生物物理