南方医科大学学报

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (1): 173-178.doi: 10.12122/j.issn.1673-4254.2024.01.20

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敲低结肠癌转移相关基因1促进RSL3诱导的结直肠癌细胞铁死亡

孙 硕，黄 鑫，李国东，张春云，卢泽梅，张伟伟，李泽彦，杨清竹   

  1. 齐齐哈尔大学生命科学与农林学院，黑龙江 齐齐哈尔 161006；抗性基因工程与寒地生物多样性保护黑龙江省重点实验室，黑龙江 齐齐哈尔 161006；哈尔滨医科大学附属第四医院普外科，黑龙江 哈尔滨 150001
  • 发布日期:2024-01-24

MACC1 knockdown enhances RSL3-induced ferroptosis in human colorectal cancer cells by inhibiting GPX4 expression

SUN Shuo, HUANG Xin, LI Guodong, ZHANG Chunyun, LU Zemei, ZHANG Weiwei, LI Zeyan, YANG Qingzhu   

  1. School of Life Science, Agriculture and Forestry, Qiqihar University, Qiqihar 161006, China; Heilongjiang Provincial Key Laboratory of Resistance Gene Engineering and Protection of Biodiversity in Cold Areas, Qiqihar 161006, China; Department of General Surgery, Fourth Affiliated Hospital of Harbin Medical University, Harbin 150001, China
  • Published:2024-01-24

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摘要： 目的 探讨结肠癌转移相关基因1（MACC1）对RAS选择性致死化合物3（RSL3）诱导的结直肠癌细胞铁死亡的影响及其分子机制。方法 体外培养结直肠癌细胞SW620，HCT116，LOVO及RKO细胞，Western blot实验检测细胞中MACC1表达；以SW620细胞为实验材料，MTT法检测不同浓度（0、2.5、5、10、20、40 μmol/L）铁死亡诱导剂RSL3以及不同浓度（0、5、10、20 μmol/L）铁死亡抑制剂 Fer-1 对 SW620 细胞存活率的影响，分析单独使用 10 μmol/L RLS3 以及 10 μmol/L RLS3 和10 μmol/L Fer-1联合作用对SW620细胞存活率的影响，并检测干扰MACC1后不同浓度RSL3对SW620细胞存活率的影响；实时定量RT-PCR和Western blot法检测不同浓度RSL3（0、2.5、5、10 μmol/L）对MACC1在mRNA和蛋白水平的影响，并检测干扰MACC1后GPX4在mRNA和蛋白水平的表达；流式细胞仪及激光共聚焦实验检测干扰MACC1后，SW620细胞中脂质过氧化 Lipid ROS 水平的变化。结果 4 种结直肠癌细胞中 SW620 细胞 MACC1 表达量最高；铁死亡诱导剂 RSL3 抑制SW620细胞的存活率，细胞存活率随RSL3浓度升高而降低，呈剂量依赖性；不同浓度的铁死亡抑制剂Fer-1对SW620细胞的存活率没有影响；与对照Ctrl组细胞存活率相比，单独使用RSL3细胞存活率降低了50%（P<0.01），而联合使用RSL3与Fer-1处理SW620细胞，细胞的存活率得到恢复（P>0.05）；不同浓度的RSL3作用于SW620细胞后，细胞中MACC1基因在mRNA 和蛋白水平被显著抑制（P<0.01），具有一定的药物浓度依赖性。siRNA 干扰 MACC1 基因表达后，增强 RSL3 对SW620细胞毒作用，并抑制细胞中GPX4的表达（P<0.01），增加细胞中Lipid ROS水平（P<0.05）。结论 MACC1通过调控GPX4影响RSL3诱导的结直肠癌细胞铁死亡。

关键词: RAS选择性致死化合物3；铁死亡；结肠癌转移相关基因1；谷胱甘肽过氧化物酶4

Abstract: Objective To investigate the effect of MACC1 on RSL3-induced ferroptosis in colorectal cancer cells and explore its molecular mechanism. Methods MACC1 expression was detected in SW620, HCT116, LOVO and RKO cells using Western blotting. The effects of different concentrations of RSL3 (an inducer of ferroptosis) or Fer-1 (an inhibitor of ferroptosis) alone, or 10 μmol/L RLS3 combined with 10 μmol/L Fer-1, on viability of SW620 cells were examined using MTT assay. The survival of SW620 cells with mRNA interference of MACC1 was analyzed following treatment with RSL3, and RT-qPCR and Western blotting were performed to detect the changes in MACC1 expressions after RSL3 treatment at different concentrations and the changes in GPX4 expression after MACC1 knockdown. Flow cytometry and laser confocal microscopy were used to analyze the changes in ROS-induced lipid peroxidation in SW620 cells after MACC1 knockdown. Results SW620 cells had the highest MACC1 expression among the 4 colorectal cancer cell lines. Treatment with RSL3 significantly inhibited the viability of SW620 cells in a dose-dependent manner, while Fer-1 did not significantly affect the survival of SW620 cells. RSL3 alone reduced SW620 cell survival by 50% (P<0.01), and the combined treatment with RSL3 and Fer-1 caused no significant changes in cell survival (P>0.05). Treatment with RSL3 concentration-dependently suppressed MACC1 expressions at both the mRNA and protein levels in SW620 cells (P<0.01). MACC1 knockdown obviously enhanced the cytotoxic effect of RSL3, inhibited the expression of GPX4, and increased ROS-induced lipid peroxidation in SW620 cells (P<0.05). Conclusion MACC1 knockdown enhances RSL3-induced ferroptosis in cultured colorectal cancer cells by inhibiting the expression of GPX4.

Key words: RAS-selective lethal 3; ferroptosis; metastasis-associated in colon cancer 1; glutathione peroxidase 4