Department of Ophthalmology, Duke University, Durham, North Carolina, USA.
Invest Ophthalmol Vis Sci. 2011 Jun 1;52(6):3567-72. doi: 10.1167/iovs.10-6448.
To investigate the interactions between microRNA-29 (miR-29), a negative regulator of extracellular matrix (ECM), and transforming growth factors (TGF)β-1 and TGFβ-2.
Changes in expression of the miR-29 family were analyzed by quantitative-PCR (Q-PCR) after treatment with TGFβ1 and TGFβ2 (1 ng/mL). TGFβ1 and TGFβ2 were evaluated at gene expression and protein levels by Q-PCR and ELISA, respectively, in human trabecular meshwork (HTM) cells transfected with miR-29b or scramble control. TGFβ1 promoter activity was analyzed using an adenovirus with the reporter SEAP. The effects of miR-29b and TGFβ2 on ECM gene expression were evaluated in cells transfected with miR-29b or scramble control and treated with TGFβ2, and the expression of ECM genes was analyzed by Q-PCR.
TGFβ2 but not TGFβ1, downregulated the three members of the miR-29 family. Overexpression of miR-29b antagonized the effects of TGFβ2 on the expression of several ECM components. MiR-29b decreased the expression of TGFβ1 at the promoter, transcript, and protein levels but had only a minor effect on the expression of active TGFβ2. The inhibition of TGFβ1 by miR-29b was partially recovered after co-transfection with a plasmid-expressing bone morphogenetic protein 1.
Results showed some level of crosstalk between TGFβs and miR-29. Specifically, the downregulation of miR-29 by TGFβ2 contributed to the induction of several ECM components by this cytokine in TM cells. This observation, together with the inhibitory effects of miR-29b on the expression of TGFβ1, suggests that the miR-29 family could play an important role in modulating TGFβs on the outflow pathway.
研究细胞外基质（ECM）负调控因子 microRNA-29（miR-29）与转化生长因子（TGF）β-1 和 TGFβ-2 之间的相互作用。
通过 TGFβ1 和 TGFβ2（1ng/mL）处理后，用定量 PCR（Q-PCR）分析 miR-29 家族的表达变化。通过 Q-PCR 和 ELISA 分别评估 TGFβ1 和 TGFβ2 在人眼小梁网（HTM）细胞中的基因表达和蛋白水平，这些细胞转染了 miR-29b 或对照 scramble。用携带 SEAP 报告基因的腺病毒分析 TGFβ1 启动子活性。在转染了 miR-29b 或对照 scramble 的细胞中，用 TGFβ2 处理后评估 miR-29b 和 TGFβ2 对 ECM 基因表达的影响，并通过 Q-PCR 分析 ECM 基因的表达。
TGFβ2 而非 TGFβ1 下调了 miR-29 家族的三个成员。miR-29b 的过表达拮抗了 TGFβ2 对几种 ECM 成分表达的影响。miR-29b 降低了 TGFβ1 在启动子、转录本和蛋白水平的表达，但对活性 TGFβ2 的表达影响较小。在用表达骨形态发生蛋白 1 的质粒共转染后，miR-29b 对 TGFβ1 的抑制作用部分恢复。
结果表明 TGFβs 和 miR-29 之间存在一定程度的串扰。具体而言，TGFβ2 下调 miR-29 有助于该细胞因子诱导 TM 细胞中几种 ECM 成分的表达。这一观察结果，加上 miR-29b 对 TGFβ1 表达的抑制作用，表明 miR-29 家族可能在调节 TGFβs 对流出途径的作用方面发挥重要作用。
