Neurology Department, Second Affiliated Hospital of Kunming Medical University, Kunming Yunnan, China.
Kunming Medical University, Kunming Yunnan, China.
Ann Clin Lab Sci. 2024 Mar;54(2):179-189.
Cerebral microbleeds (CMBs) are punctate hemorrhagic lesions within the brain parenchyma and are a classic manifestation of cerebral small vessel disease (CSVD). The primary objective of this study is to investigate the potential role of miR-4685-3p and underlying mechanisms by which miR-4685-3p modulates matrix metalloproteinase-9 (MMP9) in cerebral microvascular endothelial cell injury.
We employed high-throughput sequencing to screen for differentially expressed miRNAs in the peripheral blood of patients with CMBs and healthy controls. Employing lipopolysaccharide (LPS) to induce cellular damage, we aim to establish a model of human brain microvascular endothelial cells (hCMEC/D3) injury. We also had cells transfected with miR-4685-3p mimic and MMP9 overexpression plasmid. We utilized quantitative polymerase chain reaction (qPCR) to assess the expression levels of miR-4685-3p and performed Western blot analysis to examine MMP9 expression levels in the cells. We employed the CCK-8 assay, TUNEL assay, and tube formation assay to evaluate cellular viability, apoptotic rates, and angiogenic capabilities. Furthermore, dual-luciferase reporter assay analysis was conducted to confirm the relationship between miR-4685-3p and MMP9.
The sequencing results indicated a downregulation of miR-4685-3p in the peripheral blood of patients with CMBs. Within the context of LPS-induced injury to hCMEC/D3 cells, miR-4685-3p exhibits reduced expression, whereas MMP9 expression levels are elevated. The elevation of miR-4685-3p expression levels attenuates LPS-induced cellular apoptosis and enhances the viability and tube-forming capacity of hCMEC/D3 cells. Concomitant transfection with MMP9 overexpression constructs effectively reversed the detrimental effects of LPS on hCMEC/D3 cell integrity. We further confirmed that miR-4685-3p overexpression directly targets MMP9, leading to negative regulation of MMP9 expression.
Upregulating miR-4685-3p, which targets the MMP9 axis, mitigated LPS-induced cerebral microvascular endothelial cell injury, potentially playing a protective role in the progression of CMBs.
脑微出血（CMBs）是脑实质内点状出血性病变，是脑小血管病（CSVD）的典型表现。本研究的主要目的是探讨 miR-4685-3p 的潜在作用及其调节基质金属蛋白酶-9（MMP9）在脑微血管内皮细胞损伤中的潜在机制。
我们采用高通量测序筛选 CMB 患者和健康对照者外周血中差异表达的 miRNA。采用脂多糖（LPS）诱导细胞损伤，建立人脑微血管内皮细胞（hCMEC/D3）损伤模型。同时，用 miR-4685-3p 模拟物和 MMP9 过表达质粒转染细胞。采用实时定量聚合酶链反应（qPCR）检测 miR-4685-3p 的表达水平，采用 Western blot 分析检测细胞中 MMP9 的表达水平。采用 CCK-8 法、TUNEL 法和管形成试验评估细胞活力、凋亡率和血管生成能力。此外，还进行了双荧光素酶报告基因分析以验证 miR-4685-3p 与 MMP9 之间的关系。
测序结果显示，CMBs 患者外周血中 miR-4685-3p 表达下调。在 LPS 诱导的 hCMEC/D3 细胞损伤中，miR-4685-3p 表达降低，而 MMP9 表达水平升高。上调 miR-4685-3p 表达水平可减轻 LPS 诱导的细胞凋亡，并增强 hCMEC/D3 细胞的活力和管形成能力。同时转染 MMP9 过表达构建体可有效逆转 LPS 对 hCMEC/D3 细胞完整性的损害。我们进一步证实 miR-4685-3p 过表达可直接靶向 MMP9，从而负调控 MMP9 的表达。
上调 miR-4685-3p，靶向 MMP9 轴，减轻 LPS 诱导的脑微血管内皮细胞损伤，可能在 CMBs 的进展中发挥保护作用。
