Department of Neurology, The First Affiliated Hospital of Harbin Medical University, Harbin, China.
Oxid Med Cell Longev. 2019 Jun 2;2019:2140427. doi: 10.1155/2019/2140427. eCollection 2019.
Neurotoxicity induced by the amyloid- (A) peptide is one of the most important pathological mechanisms of Alzheimer's disease (AD). Based on accumulating evidence in AD research, both endoplasmic reticulum stress (ER stress) and alterations in the microRNA (miRNA) network contribute to the pathogenesis of the disease, making them potential therapeutic targets for AD. The present study was performed to investigate whether miR-34a and the inositol-requiring enzyme 1 (IRE1) are involved in the regulation of A-induced cytotoxicity.
Human neuroblastoma SH-SY5Y cells were treated with A1-40. Cell viability was assessed by the MTT assay. The integrity of the plasma membrane was assessed by LDH release. The expression levels of XBP1s, IRE1, p-IRE1, and Caspase-2 were detected by Western blot analysis. Spliced-XBP1 mRNA and miR-34a were detected by reverse transcription- (RT-) PCR and quantitative real-time PCR, respectively. Caspase-2 activity was measured using the Caspase-2 cellular activity assay kit. The IRE1 inhibitor (STF-083010) was used to determine the role of IRE1 on miR-34a expression. SH-SY5Y cells were transfected with miR-34a mimics to assess the role of miR-34a on the activation of Caspase-2 and the viability of A-exposed SH-SY5Y cells.
We showed that A caused concentration- and duration-dependent death of SH-SY5Y cells. The expression levels of XBP1s, p-IRE1, and Caspase-2 were increased, along with a corresponding decrease in the miR-34a levels in A-exposed SH-SY5Y cells. The IRE1 inhibitor (STF-083010) upregulated the expression of miR-34a and suppressed the activation of Caspase-2, effectively alleviating the A-induced death of SH-SY5Y cells. Transfection studies show that miR-34a mimics inhibit the expression of Caspase-2 and restore the viability of A-exposed SH-SY5Y cells.
A peptide induced downregulation of miR-34a through the activation of IRE1, which may induce cytotoxicity by targeting Caspase-2. Upregulation of miR-34a by inhibition of IRE1 has protective effects against A-induced injury in SH-SY5Y cells.
淀粉样蛋白-（A）肽诱导的神经毒性是阿尔茨海默病（AD）最重要的病理机制之一。基于 AD 研究中积累的证据，内质网应激（ER 应激）和 microRNA（miRNA）网络的改变都有助于疾病的发病机制，使它们成为 AD 的潜在治疗靶点。本研究旨在探讨 miR-34a 和肌醇需求酶 1（IRE1）是否参与调节 A 诱导的细胞毒性。
用 A1-40 处理人神经母细胞瘤 SH-SY5Y 细胞。通过 MTT 测定法评估细胞活力。通过 LDH 释放评估质膜的完整性。通过 Western blot 分析检测 XBP1s、IRE1、p-IRE1 和 Caspase-2 的表达水平。通过逆转录-（RT-）PCR 和定量实时 PCR 分别检测 spliced-XBP1 mRNA 和 miR-34a。使用 Caspase-2 细胞活性测定试剂盒测定 Caspase-2 活性。用 IRE1 抑制剂（STF-083010）来确定 IRE1 在 miR-34a 表达中的作用。用 miR-34a 模拟物转染 SH-SY5Y 细胞，以评估 miR-34a 对 Caspase-2 激活和 A 暴露的 SH-SY5Y 细胞活力的作用。
我们发现 A 导致 SH-SY5Y 细胞浓度和时间依赖性死亡。在 A 暴露的 SH-SY5Y 细胞中，XBP1s、p-IRE1 和 Caspase-2 的表达水平增加，而 miR-34a 的水平相应降低。IRE1 抑制剂（STF-083010）上调 miR-34a 的表达并抑制 Caspase-2 的激活，有效减轻了 A 诱导的 SH-SY5Y 细胞死亡。转染研究表明，miR-34a 模拟物抑制 Caspase-2 的表达并恢复 A 暴露的 SH-SY5Y 细胞活力。
A 肽通过激活 IRE1 下调 miR-34a 的表达，这可能通过靶向 Caspase-2 诱导细胞毒性。通过抑制 IRE1 上调 miR-34a 对 SH-SY5Y 细胞的 A 诱导损伤具有保护作用。
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