Luna Coralia, Li Guorong, Qiu Jianmimg, Epstein David L, Gonzalez Pedro
Department of Ophthalmology, Duke University, Durham, NC.
Mol Vis. 2009 Nov 28;15:2488-97.
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).
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.
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.
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.
研究微小RNA-29b（miR-29b）在慢性氧化应激诱导人小梁网细胞（HTM）细胞外基质（ECM）合成与沉积相关基因表达变化中的作用。
使用Affymetrix U133A2芯片通过基因阵列分析评估miR-29b在HTM细胞中诱导的基因表达变化，并通过定量聚合酶链反应（q-PCR）进行验证。使用Metacore进行通路分析。使用Psicheck荧光素酶系统评估miR-29b对三个新的假定靶标的3'-非翻译区的靶向作用。通过在40%氧气条件下孵育4-5天诱导慢性氧化应激，以在5%氧气条件下孵育的培养物作为对照。通过q-PCR分析微小RNA或基因表达的变化。通过乳酸脱氢酶释放测定细胞活力。
用miR-29b模拟物转染HTM细胞导致多种ECM成分下调，包括胶原蛋白（COL1A1、COL1A2、COL4A1、COL5A1、COL5A2、COL3A1）、层粘连蛋白γ1（LAMC1）和纤连蛋白（FBN）以及一些参与ECM沉积和重塑的基因，如富含半胱氨酸的酸性分泌蛋白/骨连接蛋白（SPARC/osteonectin）。另外三个基因，骨形态发生蛋白1（BMP1）、解聚素和金属蛋白酶12（ADAM12）和核因子κB抑制蛋白相关蛋白2（NKIRAS2），被鉴定为miR-29b的直接靶标。慢性氧化应激在两种HTM细胞系中诱导miR-29b显著下调，这与几种已知受miR-29b调控的ECM基因表达增加有关。用miR-29b模拟物转染可抑制这些基因表达的增加。在慢性氧化应激和生理氧浓度下，miR-29b均可提高细胞活力。
miR-29b负向调节小梁网（TM）细胞中参与ECM合成与沉积的多个基因的表达。miR-29b的下调可能导致慢性氧化应激条件下几种ECM基因表达增加。氧化应激诱导的ECM产生激活与miR-29b的保护作用之间的平衡可能是理解氧化损伤如何导致TM中ECM沉积增加并导致青光眼眼压升高的一个相关因素。
