Department of Biology, Urmia University, Urmia, Iran.
Solid Tumor Research Center, Cellular and Molecular Medicine Institute, Urmia University of Medical Sciences, Urmia, Iran.
Tissue Cell. 2022 Jun;76:101803. doi: 10.1016/j.tice.2022.101803. Epub 2022 Apr 21.
The intracellular endomembrane system contributes to maintaining cell homeostasis. We investigate the dynamic of exosomal and autophagy pathways in Human Umbilical Vein Cells (HUVECs) following incubation with H2O2 in vitro. Cellular senescence was induced to HUVECs using 100 µM HO. ELISA and AChE assay was used to calculate the number of exosomes. Exosomes were isolated and characterized by dynamic light scattering, flow cytometry, and SEM. Transcript and protein levels of genes involved in the exosomal and autophagy pathways were measured by real-time PCR (Q-PCR) and western blotting. Subcellular distribution of CD63 was monitored by immunofluorescence microscopy. We also measured the expression of miR-182 and miR-155 by qPCR assay. Results showed that secretion of exosomes was increased in treated cells (p < 0.05). Exosomes were confirmed by size and positive for CD63 marker. Molecular analysis of the exosomal secretory pathway has revealed a significant induction of CD63, CD81, TSAP6, Rab11, Rab27a, and Rab27b in response to HO (p < 0.05). The distribution of CD63 was increased inside treated cells. The western blotting technique revealed a significant up-regulation in Becline-1 and P62 and a significant decrease in LC3 II/I ratio in treated cells (p < 0.05)s. Concomitant with an up-regulation of common molecules in exosomes biogenesis and autophagy including Atg5, P62 and P53, expression of miR-182 decreased and miR-155 increased in cells incubated with H2O2 (p < 0.05). Data suggested an induction in the exosomal secretory pathway coincided with a block in autophagy progress, accelerating senescence, which might be targeted for the treatment of age-related diseases.
细胞内的内膜系统有助于维持细胞内稳态。我们研究了体外培养的人脐静脉内皮细胞（HUVEC）在 H2O2 孵育后，外泌体和自噬途径的动态变化。使用 100 μM HO 诱导 HUVEC 衰老。使用 ELISA 和 AChE 测定法计算外泌体的数量。通过动态光散射、流式细胞术和 SEM 分离和表征外泌体。通过实时 PCR（Q-PCR）和 Western blot 测量参与外泌体和自噬途径的基因的转录和蛋白水平。通过免疫荧光显微镜监测 CD63 的亚细胞分布。我们还通过 qPCR 测定法测量了 miR-182 和 miR-155 的表达。结果表明，处理细胞中外泌体的分泌增加（p < 0.05）。外泌体通过大小和 CD63 标志物得到确认。对外泌体分泌途径的分子分析表明，HO 诱导 CD63、CD81、TSAP6、Rab11、Rab27a 和 Rab27b 显著上调（p < 0.05）。CD63 在处理细胞内的分布增加。Western blot 技术显示 Becline-1 和 P62 显著上调，LC3 II/I 比值在处理细胞中显著降低（p < 0.05）。与外泌体生物发生和自噬中常见分子的上调一致，包括 Atg5、P62 和 P53，孵育 H2O2 的细胞中 miR-182 表达降低，miR-155 表达升高（p < 0.05）。数据表明，外泌体分泌途径的诱导与自噬进程的阻断同时发生，加速衰老，这可能成为治疗与年龄相关疾病的靶点。
