Department of Cardiology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, China.
Department of Cardiology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, China.
Life Sci. 2018 Aug 15;207:110-116. doi: 10.1016/j.lfs.2018.05.055. Epub 2018 May 31.
Endothelial-to-mesenchymal transition (EndMT) contribute to diabetic cardiac fibrosis, the underlying mechanisms are poorly understood. In the study, we aimed to investigate the role of miR-328 in EndMT mediated by high glucose (HG) and the signaling pathways implicated in human umbilical vein endothelial cells (HUVECs).
EndMT of HUVECs was determined by immunofluorescent staining and western blot of the markers CD31 and α-SMA. Real-time polymerase chain reaction was used to detect mRNA expression of miR-328 and transforming growth factor β (TGF-β). SB431542 was used to study the relation of miR-328 and TGF-β during EndMT induced by HG. Over-expression and inhibition of miR-328 were achieved by transduction of miR-328 and antagomiR-328. The effects of miR-328 on expression of type I and III collagen, p-MEK1/2, p-ERK1/2 were examined by Western blot.
The level of miR-328 was significantly up-regulated in HG-induced EndMT. MiR-328 showed the independent capability of inducing EndMT, which was not related to TGF-β, and this effect was abrogated by antagomiR-328. MiR-328 affected type I collagen in a time- and dose-dependent manner and enhanced protein expression of type I and III collagens. Further investigation displayed that a significantly higher expression of p-MEK1/2 and p-ERK1/2 in HUVECs transduced with miR-328, and a lower expression of p-MEK1/2 and p-ERK1/2 in cells transduced with antagomiR-328.
These results suggest a novel role for miR-328 in HG-induced EndMT, MEK1/2-ERK1/2 pathway is likely to be involved in the associated effects. Our findings may suggest antagomiR-328 as an alternative agent in prevention of HG-induced EndMT.
内皮细胞向间充质细胞转化（EndMT）参与糖尿病性心脏纤维化，但其潜在机制尚不清楚。本研究旨在探讨 miR-328 在高糖（HG）诱导的人脐静脉内皮细胞（HUVEC）EndMT 中的作用及其涉及的信号通路。
通过免疫荧光染色和 CD31 和 α-SMA 标志物的 Western blot 检测 HUVECs 的 EndMT。实时聚合酶链反应检测 miR-328 和转化生长因子β（TGF-β）的 mRNA 表达。SB431542 用于研究 HG 诱导的 EndMT 过程中 miR-328 和 TGF-β 的关系。通过转导 miR-328 和 antagomiR-328 实现 miR-328 的过表达和抑制。通过 Western blot 检测 miR-328 对 I 型和 III 型胶原、p-MEK1/2、p-ERK1/2 表达的影响。
HG 诱导的 EndMT 中 miR-328 水平显著上调。miR-328 具有独立诱导 EndMT 的能力，与 TGF-β无关，而 antagomiR-328 则可阻断这种作用。miR-328 以时间和剂量依赖的方式影响 I 型胶原，增强 I 型和 III 型胶原的蛋白表达。进一步研究显示，转染 miR-328 的 HUVECs 中 p-MEK1/2 和 p-ERK1/2 的表达显著升高，而转染 antagomiR-328 的细胞中 p-MEK1/2 和 p-ERK1/2 的表达降低。
这些结果表明 miR-328 在 HG 诱导的 EndMT 中具有新的作用，MEK1/2-ERK1/2 通路可能参与了相关作用。我们的发现可能表明 antagomiR-328 可作为预防 HG 诱导的 EndMT 的替代药物。
