| 余沈桐,周 汝,杨 桐,崔竹青,张 静.缺氧通过SIRT1调控结直肠癌细胞自噬[J].,2019,19(13):2438-2443 |
| 缺氧通过SIRT1调控结直肠癌细胞自噬 |
| Hypoxia Promotes Autophagy in Colorectal Cancer Cells through SIRT1 |
| 投稿时间：2018-12-27 修订日期：2019-01-23 |
| DOI：10.13241/j.cnki.pmb.2019.13.008 |
| 中文关键词: 结直肠癌 缺氧 自噬 SIRT1 |
| 英文关键词: Colorectal cancer Hypoxia Autophagy SIRT1 |
| 基金项目:肿瘤生物学国家重点实验室自主研究课题（CBSKL2017Z21）；国家自然科学基金项目（81572545，81372783） |
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| 中文摘要: |
| 摘要 目的：探究缺氧调控结直肠癌细胞自噬的分子机制。方法：分别在常氧及缺氧（1%氧气浓度）条件下处理细胞，western blot检测细胞内沉默信息调节因子1（Silencing Information Regulator 1，SIRT1）及自噬相关标志分子的表达情况；慢病毒转染构建SIRT1稳定过表达或敲减细胞株，利用透射电镜观察细胞内自噬体形成的情况；使用mRFP-GFP-LC3双标腺病毒感染细胞，在激光扫描共聚焦显微镜下观察细胞自噬流的进展。结果：Western blot结果显示，缺氧条件下，HCT116及SW480细胞内SIRT1的表达水平随着缺氧时间的延长而降低，自噬特异性底物p62蛋白水平降低且LC3-I/II转换增加；与对照组相比，SIRT1过表达细胞内自噬特异性底物p62的表达水平升高而LC3-I/II转换受到抑制；相反在SIRT1敲减细胞内，p62的表达水平降低而LC3-I/II转换进一步促进。透射电镜结果发现SIRT1过表达后，细胞内自噬溶酶体形成减少、自噬体数量增多；激光共聚焦结果显示，SIRT1过表达细胞内绿色荧光淬灭减少、自噬体与自噬溶酶体的融合收到明显抑制，说明SIRT1通过抑制自噬溶酶体的形成，阻断自噬流的进展。结论：缺氧通过抑制SIRT1的表达促进结直肠癌的细胞自噬。 |
| 英文摘要: |
| ABSTRACT Objective: To explore regulatory mechanisms of autophagy in colorectal cancer cells under hypoxia. Methods: Colorectal cells were exposed to normoxia and hypoxia (1% O2), respectively. Western blot analysis was used to detect the expressions of Silencing Information Regulator 1 (SIRT1) and autophagy-related biomarkers. Lentivirus infection was used to obtained colorectal cells with stable SIRT1 overexpression or knockdown. Transmission electron microscope (TEM) was exploited to detect intracellular autophagosomes. After tumor cells were infected with adenovirus containing mRFP-GFP dual-labelled LC3, autophagy flux were detected under laser scanning confocal microscopy. Results: Western blots revealed that the expression of SIRT1 was decreased with the extension of hypoxic exposure time in HCT116 and SW480 cells. Besides, autophagy specific substrate p62 was declined and the transition of LC3-I/II was enhanced. Compared with control group, SIRT1-overexpressed SW480 cells exhibited increased expression of p62 and decreased transition of LC3-I/II. Conversely, in SIRT1-knockdowned HCT116 cells, the expression of p62 was decreased while the transition of LC3-I/II was further increased. The results from TEM observation indicated that the overexpression of SIRT1 decreased the number of autolysosomes while increased the number of autophagosomes. Moreover, confocal results revealed that in SIRT1-overexpressed cells, the quenching of green fluorescence was decreased and the fusion of autophagosomes and lysosomes was blocked, which suggested that SIRT1 inhibited the formation of autolysosomes and blocked the autophagy flux. Conclusion: Hypoxia promoted autophagy in colorectal cancer cells through inhibiting the expression of SIRT1. |
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