A large-scale RNA interference screen identifies genes that regulate autophagy at different stages.
A large-scale RNA interference screen identifies genes that regulate autophagy at different stages.
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DOI：
10.1038/s41598-018-21106-5
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发表时间：
2018-02-12
影响因子：
4.6
通讯作者：
Sheng Z
中科院分区：
文献类型：
--
作者：
Guo S;Pridham KJ;Virbasius CM;He B;Zhang L;Varmark H;Green MR;Sheng Z
失调的自噬是癌症的发病机制和治疗发展的核心。然而，自噬在癌症中是如何调节的还没有很好的理解，调节癌症自噬的基因也没有完全确定。为了更深入地了解癌症中的自噬调控，我们使用单丹酰尸胺染色在K562人类慢性髓性白血病细胞中进行了大规模的RNA干扰筛选，这是一种自噬检测方法，相当于自噬标记物LC 3B的免疫印迹或GFP-LC 3B的荧光显微镜。通过偶联单丹酰尸胺染色与荧光激活细胞分选，我们成功地分离出自噬K562细胞，我们确定了336短发夹RNA。经过Cyto-ID荧光分光光度法、LC 3B免疫印迹法和定量RT-PCR等方法的验证，共鉴定出82个自噬调控基因。其中20个基因已被报道，其余62个候选基因是新的自噬介质。生物信息学分析表明，大多数候选基因参与调节自噬的分子途径，而不是直接参与自噬过程。进一步的自噬通量测定显示，57个自噬调节基因抑制自噬起始，而21个候选基因促进自噬成熟。我们的RNA干扰筛选确定了在不同阶段调节自噬的基因，这有助于解码癌症中的自噬调节，并为开发癌症自噬相关疗法提供了新的途径。
Dysregulated autophagy is central to the pathogenesis and therapeutic development of cancer. However, how autophagy is regulated in cancer is not well understood and genes that modulate cancer autophagy are not fully defined. To gain more insights into autophagy regulation in cancer, we performed a large-scale RNA interference screen in K562 human chronic myeloid leukemia cells using monodansylcadaverine staining, an autophagy-detecting approach equivalent to immunoblotting of the autophagy marker LC3B or fluorescence microscopy of GFP-LC3B. By coupling monodansylcadaverine staining with fluorescence-activated cell sorting, we successfully isolated autophagic K562 cells where we identified 336 short hairpin RNAs. After candidate validation using Cyto-ID fluorescence spectrophotometry, LC3B immunoblotting, and quantitative RT-PCR, 82 genes were identified as autophagy-regulating genes. 20 genes have been reported previously and the remaining 62 candidates are novel autophagy mediators. Bioinformatic analyses revealed that most candidate genes were involved in molecular pathways regulating autophagy, rather than directly participating in the autophagy process. Further autophagy flux assays revealed that 57 autophagy-regulating genes suppressed autophagy initiation, whereas 21 candidates promoted autophagy maturation. Our RNA interference screen identified genes that regulate autophagy at different stages, which helps decode autophagy regulation in cancer and offers novel avenues to develop autophagy-related therapies for cancer.
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DOI：
10.1073/pnas.1321207111
发表时间：
2014-06-10
影响因子：
11.1
作者：
Ivatt, Rachael M.;Sanchez-Martinez, Alvaro;Whitworth, Alexander J.
通讯作者：
Whitworth, Alexander J.
影响因子：
15.9
作者：
Bellodi, Cristian;Lidonnici, Maria Rosa;Calabretta, Bruno
通讯作者：
Calabretta, Bruno
影响因子：
8
作者：
Iwamaru, A.;Kondo, Y.;Kondo, S.
通讯作者：
Kondo, S.
DOI：
10.1007/978-1-4939-4011-0_9
发表时间：
2016-01-01
期刊：
CHRONIC MYELOID LEUKEMIA: METHODS AND PROTOCOLS
影响因子：
--
作者：
Guo, Sujuan;Pridham, Kevin J.;Sheng, Zhi
通讯作者：
Sheng, Zhi
影响因子：
14.9
作者：
Franceschini A;Szklarczyk D;Frankild S;Kuhn M;Simonovic M;Roth A;Lin J;Minguez P;Bork P;von Mering C;Jensen LJ
通讯作者：
Jensen LJ