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Effects of metal nanoparticles on tight junction-associated proteins via HIF-1α/miR-29b/MMPs pathway in human epidermal keratinocytes
Particle and Fibre Toxicology ( IF 10 ) Pub Date : 2021-03-19 , DOI: 10.1186/s12989-021-00405-2
Jiali Yuan 1 , Yue Zhang 1 , Yuanbao Zhang 1 , Yiqun Mo 1 , Qunwei Zhang 1
Affiliation  

The increasing use of metal nanoparticles in industry and biomedicine raises the risk for unintentional exposure. The ability of metal nanoparticles to penetrate the skin ranges from stopping at the stratum corneum to passing below the dermis and entering the systemic circulation. Despite the potential health risks associated with skin exposure to metal nanoparticles, the mechanisms underlying the toxicity of metal nanoparticles on skin keratinocytes remain unclear. In this study, we proposed that exposure of human epidermal keratinocytes (HaCaT) to metal nanoparticles, such as nickel nanoparticles, dysregulates tight-junction associated proteins by interacting with the HIF-1α/miR-29b/MMPs axis. We performed dose-response and time-response studies in HaCaT cells to observe the effects of Nano-Ni or Nano-TiO2 on the expression and activity of MMP-2 and MMP-9, and on the expression of tight junction-associated proteins, TIMP-1, TIMP-2, miR-29b, and HIF-1α. In the dose-response studies, cells were exposed to 0, 10, or 20 μg/mL of Nano-Ni or Nano-TiO2 for 24 h. In the time-response studies, cells were exposed to 20 μg/mL of Nano-Ni for 12, 24, 48, or 72 h. After treatment, cells were collected to either assess the expression of mRNAs and miR-29b by real-time PCR or to determine the expression of tight junction-associated proteins and HIF-1α nuclear accumulation by Western blot and/or immunofluorescent staining; the conditioned media were collected to evaluate the MMP-2 and MMP-9 activities by gelatin zymography assay. To further investigate the mechanisms underlying Nano-Ni-induced dysregulation of tight junction-associated proteins, we employed a HIF-1α inhibitor, CAY10585, to perturb HIF-1α accumulation in one experiment, and transfected a miR-29b-3p mimic into the HaCaT cells before Nano-Ni exposure in another experiment. Cells and conditioned media were collected, and the expression and activities of MMPs and the expression of tight junction-associated proteins were determined as described above. Exposure of HaCaT cells to Nano-Ni resulted in a dose-dependent increase in the expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 and the activities of MMP-2 and MMP-9. However, exposure of cells to Nano-TiO2 did not cause these effects. Nano-Ni caused a dose-dependent decrease in the expression of miR-29b and tight junction-associated proteins, such as ZO-1, occludin, and claudin-1, while Nano-TiO2 did not. Nano-Ni also caused a dose-dependent increase in HIF-1α nuclear accumulation. The time-response studies showed that Nano-Ni caused significantly increased expressions of MMP-2 at 24 h, MMP-9 at 12, 24, and 48 h, TIMP-1 from 24 to 72 h, and TIMP-2 from 12 to 72 h post-exposure. The expression of miR-29b and tight junction-associated proteins such as ZO-1, occludin, and claudin-1 decreased as early as 12 h post-exposure, and their levels declined gradually over time. Pretreatment of cells with a HIF-1α inhibitor, CAY10585, abolished Nano-Ni-induced miR-29b down-regulation and MMP-2/9 up-regulation. Introduction of a miR-29b-3p mimic into HaCaT cells by transfection before Nano-Ni exposure ameliorated Nano-Ni-induced increased expression and activity of MMP-2 and MMP-9 and restored Nano-Ni-induced down-regulation of tight junction-associated proteins. Our study herein demonstrated that exposure of human epidermal keratinocytes to Nano-Ni caused increased HIF-1α nuclear accumulation and increased transcription and activity of MMP-2 and MMP-9 and down-regulation of miR-29b and tight junction-associated proteins. Nano-Ni-induced miR-29b down-regulation was through Nano-Ni-induced HIF-1α nuclear accumulation. Restoration of miR-29b level by miR-29b-3p mimic transfection abolished Nano-Ni-induced MMP-2 and MMP-9 activation and down-regulation of tight junction-associated proteins. In summary, our results demonstrated that Nano-Ni-induced dysregulation of tight junction-associated proteins in skin keratinocytes was via HIF-1α/miR-29b/MMPs pathway.

中文翻译:

金属纳米粒子通过HIF-1α/miR-29b/MMPs通路对人表皮角质形成细胞紧密连接相关蛋白的影响

在工业和生物医学中越来越多地使用金属纳米粒子增加了意外接触的风险。金属纳米粒子穿透皮肤的能力范围从停留在角质层到穿过真皮下方进入体循环。尽管皮肤暴露于金属纳米颗粒存在潜在的健康风险,但金属纳米颗粒对皮肤角质形成细胞的毒性机制仍不清楚。在这项研究中,我们提出将人表皮角质形成细胞 (HaCaT) 暴露于金属纳米颗粒,例如镍纳米颗粒,通过与 HIF-1α/miR-29b/MMPs 轴相互作用来失调紧密连接相关的蛋白质。我们在 HaCaT 细胞中进行了剂量反应和时间反应研究,以观察 Nano-Ni 或 Nano-TiO2 对 MMP-2 和 MMP-9 表达和活性的影响,以及对紧密连接相关蛋白表达的影响, TIMP-1、TIMP-2、miR-29b 和 HIF-1α。在剂量反应研究中,细胞暴露于 0、10 或 20 μg/mL 的纳米镍或纳米二氧化钛 24 小时。在时间响应研究中,细胞暴露于 20 μg/mL 的纳米镍 12、24、48 或 72 小时。处理后,收集细胞以通过实时 PCR 评估 mRNA 和 miR-29b 的表达,或通过蛋白质印迹和/或免疫荧光染色确定紧密连接相关蛋白的表达和 HIF-1α 核积累;收集条件培养基以通过明胶酶谱测定评估MMP-2和MMP-9活性。为了进一步研究纳米镍诱导的紧密连接相关蛋白失调的机制,我们在一项实验中采用了 HIF-1α 抑制剂 CAY10585 来扰乱 HIF-1α 的积累,并将 miR-29b-3p 模拟物转染到在另一个实验中,纳米镍暴露前的 HaCaT 细胞。收集细胞和条件培养基,如上所述测定 MMP 的表达和活性以及紧密连接相关蛋白的表达。HaCaT 细胞暴露于 Nano-Ni 导致 MMP-2、MMP-9、TIMP-1 和 TIMP-2 的表达以及 MMP-2 和 MMP-9 的活性呈剂量依赖性增加。然而,将细胞暴露于纳米二氧化钛不会导致这些影响。Nano-Ni 引起 miR-29b 和紧密连接相关蛋白(如 ZO-1)表达的剂量依赖性降低,occludin 和 claudin-1,而 Nano-TiO2 没有。Nano-Ni 还导致 HIF-1α 核积累的剂量依赖性增加。时间响应研究表明,纳米镍使 MMP-2 在 24 h、MMP-9 在 12、24 和 48 h、TIMP-1 在 24-72 h 和 TIMP-2 在 12-72 h 的表达显着增加。暴露后 72 小时。miR-29b 和紧密连接相关蛋白(如 ZO-1、occludin 和 claudin-1)的表达早在暴露后 12 小时就下降,并且它们的水平随着时间的推移逐渐下降。用 HIF-1α 抑制剂 CAY10585 预处理细胞,消除了纳米镍诱导的 miR-29b 下调和 MMP-2/9 上调。在纳米镍暴露前通过转染将 miR-29b-3p 模拟物引入 HaCaT 细胞,改善纳米镍诱导的 MMP-2 和 MMP-9 表达和活性增加,并恢复纳米镍诱导的紧密连接下调-相关蛋白。我们在此的研究表明,人表皮角质形成细胞暴露于纳米镍会导致 HIF-1α 核积累增加,MMP-2 和 MMP-9 的转录和活性增加,以及 miR-29b 和紧密连接相关蛋白的下调。纳米镍诱导的 miR-29b 下调是通过纳米镍诱导的 HIF-1α 核积累。通过 miR-29b-3p 模拟物转染恢复 miR-29b 水平消除了纳米镍诱导的 MMP-2 和 MMP-9 激活和紧密连接相关蛋白的下调。总之,
更新日期:2021-03-21
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