Virginia Tech Carilion Research Institute, Roanoke, VA, 24016, United States.
Graduate Program in Translational Biology, Medicine, and Health, Virginia Tech, Blacksburg, VA, 24061, United States.
Sci Rep. 2018 Feb 12;8(1):2822. doi: 10.1038/s41598-018-21106-5.
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 identifies 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.
自噬失调是癌症发病机制和治疗开发的核心。然而，自噬在癌症中是如何被调节的还不太清楚，调节癌症自噬的基因也没有完全定义。为了更深入地了解癌症中的自噬调节，我们使用单丹磺酰尸胺染色法在 K562 人慢性髓系白血病细胞中进行了大规模的 RNA 干扰筛选，这是一种与自噬标志物 LC3B 的免疫印迹或 GFP-LC3B 的荧光显微镜相当的自噬检测方法。通过将单丹磺酰尸胺染色与荧光激活细胞分选相结合，我们成功分离出自噬 K562 细胞，在这些细胞中我们鉴定出 336 个短发夹 RNA。在用 Cyto-ID 荧光分光光度法、LC3B 免疫印迹和定量 RT-PCR 进行候选验证后，鉴定出 82 个基因作为自噬调节基因。其中 20 个基因以前有报道过，其余 62 个候选基因是新的自噬介体。生物信息学分析显示，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表明，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 干扰筛选鉴定出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，并为开发癌症相关的自噬治疗提供新的途径。
