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.

中文翻译：

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MiR-32-5p通过抑制DUSP1影响高葡萄糖诱导的心脏成纤维细胞增殖和表型改变。

目前的研究旨在研究miR-32-5p对高糖诱导的心脏成纤维细胞（CFs）的影响。我们还旨在确定参与DUSP1表达调控的潜在机制。将人CFs用miR-32-5p抑制剂转染或模拟，并用正常浓度或高浓度的葡萄糖进行处理。通过检查人CF中的波形蛋白，纤连蛋白（FN）和α-肌动蛋白的表达，进行流式细胞仪分析以鉴定心脏成纤维细胞。进行了qRT-PCR和Western blot分析，以确认miR-32-5p，DUSP1和心脏纤维化相关蛋白的表达。通过使用MTT测定法评估CF的增殖。进行了免疫细胞荧光染色测定法，以确定α-SMA的蛋白质水平并研究人CFs的表型变化程度。miR-32-5p和DUSP1之间的特定关系通过双重萤光素酶报告基因分析进行了研究。用流式细胞仪和膜联蛋白V-FITC和碘化丙啶（PI）染色法测量细胞凋亡率。荧光素酶报告基因测定表明miR-32-5p可以直接靶向DUSP1。高葡萄糖水平导致miR-32-5p的过表达，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。