ABSTRACT

Background

The number of neutrophils is significantly reduced in myelodysplastic syndrome (MDS), but the molecular basis remains unclear. We recently found that miR-34a was significantly increased in MDS neutrophils. Therefore, this study aims to clarify the effects of aberrant miR-34a expression on neutrophil counts.

Methods

miR-34a mimics/inhibitor transfection were performed in neutrophil-like differentiated HL60 (dHL60) cells, and a FACSCalibur flow cytometer was used to measure ROS production and apoptosis. In addition, the Cdc42-WASP-Arp2/3 pathway inhibitor (ML141) and activator (CN02) treated the dHL60 cells, and then ROS production, apoptosis and related proteins expression were detected. And, luciferase reporter assay to verify the relationship of miR-34a and the Cdc42-WASP-Arp2/3 pathway.

Results

overexpression of miR-34a could induce ROS production and apoptosis, decrease the expression levels of DOCK8, p-WASP, WASP, Arp2, Arp3, and increase F-actin’s expression. Meanwhile, knockdown of miR-34a could decrease ROS production and apoptosis, increase the expression of DOCK8, p-WASP, WASP, Arp2, Arp3, and decrease F-actin’s expression. Immunofluorescence staining showed aberrant miR-34a and Cdc42-WASP-Arp2/3 pathway could induce F-actin membrane transfer. Luciferase reporter assay indicated that DOCK8 was a direct target gene of miR-34a.

Conclusion

These data indicates miR-34a may induce neutrophil apoptosis by regulating Cdc42-WASP-Arp2/3 pathway-mediated F-actin remodeling and ROS production.

中文翻译：

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miR-34a 通过调节 Cdc42-WASP-Arp2/3 通路介导的 F-肌动蛋白重塑和 ROS 产生诱导中性粒细胞凋亡

摘要

背景

骨髓增生异常综合征 (MDS) 中中性粒细胞的数量显着减少，但分子基础仍不清楚。我们最近发现 miR-34a 在 MDS 中性粒细胞中显着增加。因此，本研究旨在阐明异常 miR-34a 表达对中性粒细胞计数的影响。

方法

在中性粒细胞样分化的 HL60 (dHL60) 细胞中进行 miR-34a 模拟物/抑制剂转染，并使用 FACSCalibur 流式细胞仪测量 ROS 产生和细胞凋亡。此外，Cdc42-WASP-Arp2/3通路抑制剂（ML141）和激活剂（CN02）对dHL60细胞进行处理，然后检测ROS的产生、凋亡和相关蛋白的表达。并且，荧光素酶报告基因检测验证了 miR-34a 和 Cdc42-WASP-Arp2/3 通路的关系。

结果

过表达 miR-34a 可诱导 ROS 产生和细胞凋亡，降低 DOCK8、p-WASP、WASP、Arp2、Arp3 的表达水平，并增加 F-actin 的表达。同时，敲低 miR-34a 可以减少 ROS 的产生和细胞凋亡，增加 DOCK8、p-WASP、WASP、Arp2、Arp3 的表达，并降低 F-actin 的表达。免疫荧光染色显示异常的 miR-34a 和 Cdc42-WASP-Arp2/3 通路可诱导 F-actin 膜转移。荧光素酶报告基因检测表明 DOCK8 是 miR-34a 的直接靶基因。

结论

这些数据表明 miR-34a 可能通过调节 Cdc42-WASP-Arp2/3 通路介导的 F-肌动蛋白重塑和 ROS 产生来诱导中性粒细胞凋亡。