Department of Rehabilitation, Shenzhen Longgang Central Hospital, Shenzhen, 518116, China.
Department of Rehabilitation, The First Affiliated Hospital of Harbin Medical University, Harbin, 150001, China.
Cell Mol Biol (Noisy-le-grand). 2020 Oct 31;66(7):12-17.
The current research aimed to investigate the effect of miR-7 targeting matrix metalloproteinase 14 (MMP-14) on homocysteine (Hcy)-induced rat cerebral artery vascular smooth muscle cells (VSMCs) proliferation, migration and inflammatory factor expression and its possible mechanism. The expression of miR-7 and MMP-14 in Hcy-induced VSMCs were detected by real-time fluorescent quantitative PCR (RT-qPCR) and Western blot. Methyl Thiazolyl Tetrazolium (MTT) method, Transwell assays and enzyme-linked immunosorbent assay (ELISA) were performed to detect the effect of miR-7 and MMP-14 expression on the proliferation and migration, as well as interleukin 6 (IL-6) and tumor necrosis factor ɑ (TNF-ɑ) expression of Hcy-induced VSMCs. The interaction between miR-7 and MMP-14 was detected by dual-luciferase reporter gene assay. Western blot was applied to analyse the effects of miR-7 and MMP-14 expression on the Toll-like receptor (TLR4)/nuclear transcription factor-KB (NF-κB) signaling pathway. The results showed that after induced by Hcy, the expression of miR-7 in VSMCs was significantly reduced, the expression of MMP-14 was significantly increased, and the cell viability, the number of migrating cells, IL-6 and TNF-ɑ expression were significantly increased (P<0.05). After overexpression of miR-7, the viability, migration cell numbers, IL-6 and TNF-ɑ expression of Hcy-induced VSMCs were significantly reduced (P<0.05). miR-7 directly binds to MMP-14 and negatively regulates the expression of MMP-14. After overexpression of miR-7, the levels of TLR4 and p-NF-κB p65 in VSMCs were significantly reduced (P<0.05); overexpression of MMP-14 could reduce the effect of miR-7 overexpression on TLR4 and p-NF-κB p65 expression in VSMCs (P<0.05). Overexpression of MMP-14 and/or activation of the TLR4/NF-κB signaling pathway could reverse the effect of miR-7 overexpression on the proliferation, migration and IL-6 and TNF-ɑ expression of Hcy-induced VSMCs (P<0.05). It is concluded that miR-7 can inhibit Hcy-induced rat cerebral artery VSMCs proliferation, migration, and inflammatory factor expression by targeting the regulation of MMP-14 expression and inhibiting the activation of the TLR4/NF-κB signaling pathway.
本研究旨在探讨 miR-7 靶向基质金属蛋白酶 14（MMP-14）对同型半胱氨酸（Hcy）诱导的大鼠脑动脉血管平滑肌细胞（VSMCs）增殖、迁移和炎症因子表达的影响及其可能的机制。采用实时荧光定量 PCR（RT-qPCR）和 Western blot 检测 Hcy 诱导的 VSMCs 中 miR-7 和 MMP-14 的表达。采用甲基噻唑基四唑（MTT）法、Transwell 实验和酶联免疫吸附试验（ELISA）检测 miR-7 和 MMP-14 表达对 Hcy 诱导的 VSMCs 增殖和迁移以及白细胞介素 6（IL-6）和肿瘤坏死因子 ɑ（TNF-ɑ）表达的影响。采用双荧光素酶报告基因检测 miR-7 与 MMP-14 的相互作用。Western blot 分析 miR-7 和 MMP-14 表达对 Toll 样受体（TLR4）/核转录因子-KB（NF-κB）信号通路的影响。结果显示，Hcy 诱导后，VSMCs 中 miR-7 的表达明显降低，MMP-14 的表达明显升高，细胞活力、迁移细胞数、IL-6 和 TNF-ɑ 的表达明显升高（P<0.05）。过表达 miR-7 后，Hcy 诱导的 VSMCs 的活力、迁移细胞数、IL-6 和 TNF-ɑ 的表达明显降低（P<0.05）。miR-7 可直接结合 MMP-14，负调控 MMP-14 的表达。过表达 miR-7 后，VSMCs 中 TLR4 和 p-NF-κB p65 的水平明显降低（P<0.05）；过表达 MMP-14 可降低 VSMCs 中 TLR4 和 p-NF-κB p65 表达受 miR-7 过表达的影响（P<0.05）。过表达 MMP-14 和/或激活 TLR4/NF-κB 信号通路可逆转 miR-7 过表达对 Hcy 诱导的 VSMCs 增殖、迁移和 IL-6 和 TNF-ɑ 表达的影响（P<0.05）。综上所述，miR-7 可通过靶向调控 MMP-14 表达抑制 TLR4/NF-κB 信号通路的激活，抑制 Hcy 诱导的大鼠脑动脉 VSMCs 的增殖、迁移和炎症因子表达。
