Department of Cardiology, Children's Hospital, Zhejiang University School of Medicine, No. 3333 Binsheng Road, Binjiang District, Hangzhou, 310051, Zhejiang, China.
Children's Heart Center, Sichuan Provincial Hospital for Women and Children, Chengdu, 610045, Sichuan, China.
BMC Mol Biol. 2019 Aug 22;20(1):21. doi: 10.1186/s12867-019-0135-x.
The current study aimed to investigate the effects of miR-32-5p on cardiac fibroblasts (CFs) that were induced with high levels of glucose; we also aimed to identify the potential mechanisms involved in the regulation of DUSP1 expression.
Human CFs were transfected with a miR-32-5p inhibitor or mimic and were treated with a normal concentration or a high concentration of glucose. Flow cytometry analysis was performed to identify cardiac fibroblasts by examining vimentin, fibronectin (FN) and α-actin expression in human CFs. qRT-PCR and western blot assays were performed to confirm the expression of miR-32-5p, DUSP1 and cardiac fibrosis relevant proteins. The proliferation of CFs was assessed by using MTT assay. An immunocytofluorescent staining assay was performed to determine the protein level of α-SMA and to investigate the degree of phenotypic changes in human CFs. The specific relationship between miR-32-5p and DUSP1 was investigated by a dual luciferase reporter assay. Cell apoptosis rates were measured with flow cytometry and the annexin V-FITC and propidine iodide (PI) staining method.
A luciferase reporter assay indicated that miR-32-5p could directly target DUSP1. High glucose levels resulted in the overexpression of miR-32-5p, which downregulated DUSP1 expression. Both the upregulation of miR-32-5p and the downregulation of DUSP1 promoted cell apoptosis, proliferation and phenotypic changes in human CFs.
All findings in this study provide further evidence for the positive effects of miR-32-5p on cell proliferation and the phenotypic changes in CFs by inhibiting DUSP1 expression, and reveal that miR-32-5p could serve as prognostic diagnostic target for cardiac fibrosis.
本研究旨在探讨高糖诱导的心肌成纤维细胞（CFs）中 miR-32-5p 的作用，并鉴定其调控 DUSP1 表达的潜在机制。
用 miR-32-5p 抑制剂或模拟物转染人 CFs，并用正常浓度或高浓度葡萄糖处理。通过检查人 CFs 中波形蛋白、纤连蛋白（FN）和α-肌动蛋白的表达，用流式细胞术分析鉴定 CFs。用 qRT-PCR 和 Western blot 检测 miR-32-5p、DUSP1 和与心肌纤维化相关的蛋白的表达。用 MTT 检测 CFs 的增殖。用免疫细胞荧光染色法检测α-SMA 的蛋白水平，并研究人 CFs 表型变化的程度。通过双荧光素酶报告基因检测法研究 miR-32-5p 与 DUSP1 之间的特异性关系。用流式细胞术测量细胞凋亡率，并用 Annexin V-FITC 和碘化丙啶（PI）染色法检测。
荧光素酶报告基因检测表明，miR-32-5p 可以直接靶向 DUSP1。高糖水平导致 miR-32-5p 过表达，从而下调 DUSP1 的表达。miR-32-5p 的上调和 DUSP1 的下调均促进人 CFs 的细胞凋亡、增殖和表型变化。
本研究中的所有发现进一步证明了 miR-32-5p 通过抑制 DUSP1 的表达对 CFs 增殖和表型变化的积极作用，并表明 miR-32-5p 可作为心肌纤维化的预后诊断靶标。
