南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (1): 52-59.doi: 10.12122/j.issn.1673-4254.2023.01.07

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ANP32A沉默体外抑制结直肠癌的生长、侵袭和迁移：基于AKT信号通路活性的下降

丁虹芳，李肖娟，周璐炜，崔 智，蒙海德，王 娟   

  1. 桂林医学院基础医学院，药学院，生物技术学院，广西 桂林 541199
  • 出版日期:2023-01-20 发布日期:2023-02-22

Silenced ANP32A inhibits the growth, invasion and migration of colorectal cancer in vitro via the inactivation of AKT pathway

DING Hongfang, LI Xiaojuan, ZHOU Luwei, CUI Zhi, MENG Haide, WANG Juan   

  1. Faculty of Basic Medical Sciences, School of Pharmacy, School of Intelligent Medicine and Biotechnology, of Guilin Medical College, Guilin 541199, China
  • Online:2023-01-20 Published:2023-02-22

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摘要： 目的 探讨ANP32A基因下调对结直肠癌细胞侵袭迁移的作用与AKT 信号通路活性的相关性。方法 采用慢病毒转染技术构建结直肠癌细胞 HCT116和SW480 sh-NC和 sh-ANP32A的稳定细胞株，MTT检测24、48及72 h时ANP32A敲低对细胞增殖的影响，Western blot检测ANP32A敲低对 AKT的活性的影响；应用AKT的激活剂（SC79）和抑制剂（MK2206）干预HCT116及 SW480 细胞，MTT 检测 24、48、72 及 96 h 时 SC79 和 MK2206 对结直肠癌细胞增殖的影响，Transwell 小室观察 SC79 和MK2206对结直肠癌细胞侵袭迁移的作用；然后用SC79和MK2206干预上述两种结直肠癌细胞的sh-NC 和 sh-ANP32A稳定细胞株，将每种细胞分为 sh-NC 对照组，sh-NC SC79，sh-NC MK2206，sh-ANP32A 对照组，sh-ANP32A SC79，sh-ANP32AMK2206 六个组，细胞划痕分析细胞迁移能力，Transwell 小室分析细胞侵袭能力，WB 检测SC79和MK2206对shANP32A细胞侵袭迁移相关因子metadherin（MTDH）的影响。结果 与对照组（sh-NC）相比，24、48及72 h检测的sh-ANP32A组结直肠癌细胞的增殖活力均被明显抑制（P<0.01），同时 ANP32A 的下调能抑制 HCT116 和 SW480 细胞内 AKT 的活性（P<0.01）；AKT抑制剂MK2206能抑制大肠癌细胞的增殖及侵袭迁移（P<0.05），而AKT激活剂SC79 虽然对细胞增殖影响较小，但是能显著促进大肠癌的侵袭转移（P<0.01）；在ANP32A下调的细胞中，与sh-ANP32A对照组相比，AKT抑制剂MK2206能加强ANP32A下调对结直肠癌细胞增殖和侵袭的抑制作用（P<0.05），同时增强ANP32A表达下调对MTDH表达的抑制作用，AKT激活剂SC79能部分恢复大肠癌细胞因ANP32A下调导致的侵袭迁移抑制（P<0.01），且减弱ANP32A表达下调对MTDH表达的抑制作用。结论 ANP32A表达下调抑制结直肠癌细胞的侵袭迁移作用可能与AKT 信号通路活性抑制有关。

关键词: ANP32A；AKT；侵袭；迁移；结直肠癌

Abstract: Objective To investigate the effect of ANP32A silencing on invasion and migration of colon cancer cells and the influence of the activity of AKT signaling pathway on this effect. Methods Colorectal cancer HCT116 and SW480 were transfected with a small interfering RNA targeting ANP32A via a lentiviral vector. At 24, 48 and 72 h after the transfection, the changes in cell proliferation and AKT activity in the cells were detected using MTT assay and Western blotting, respectively. HCT116 and SW480 cells were treated with the AKT agonist SC79 or its inhibitor MK2206 for 24, 48, 72 and 96 h, and the changes in cell migration and invasion ability were analyzed using Transwell chamber assay and cell proliferation was assessed using MTT assay. The effects of SC79 and MK2206 on migration and invasion abilities of HCT116 and SW480 cells with or without ANP32A silencing were examined using wound healing and Transwell chamber assays, and the changes in the expression of metadherin (MTDH), a factor associated with cells invasion and migration, was detected with Western blotting. Results Lentivirus-mediated ANP32A silencing significantly down-regulated the activity of AKT and inhibited the proliferation of both HCT116 and SW480 cells (P<0.01). The application of AKT inhibitor MK2206 obviously inhibited the proliferation, invasion and migration of the colorectal cancer cells (P<0.05), while the AKT agonist SC79 significantly promoted the invasion and migration of the cells (P<0.01). In HCT116 and SW480 cells with ANP32A silencing, treatment with MK2206 strongly enhanced the inhibitory effects of ANP32A silencing on cell invasion and migration (P<0.05) and the expression of MTDH, while SC79 partially reversed these inhibitory effects (P<0.01). Conclusion ANP32A silencing inhibits invasion and migration of colorectal cancer cells possibly by inhibiting the activation of the AKT signaling pathway

Key words: ANP32A; AKT; invasion; migration; colon cancer