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中华肺部疾病杂志(电子版) ›› 2025, Vol. 18 ›› Issue (03) : 362 -368. doi: 10.3877/cma.j.issn.1674-6902.2025.03.004

论著

亚麻木酚素通过circRNA HIPK3影响非小细胞肺癌A549 细胞凋亡及铁死亡的机制研究
周玲1, 肖颖2,(), 李秋诗1, 陈兆毅1, 李琪3, 吴园明1   
  1. 1. 430080 武汉,武汉市普仁医院呼吸与危重症学科
    2. 430080 武汉,武汉市普仁医院血液内科
    3. 430080 武汉,武汉科技大学临床第四学院内科教研室
  • 收稿日期:2025-03-25 出版日期:2025-06-25
  • 通信作者: 肖颖
  • 基金资助:
    中国金属学会治金安全与健康分会健康卫生科研项目(jkws202411)

Mechanism of secoisolariciresinol diglucoside influencing apoptosis and ferroptosis in non-small cell lung cancer A549 cells through circRNA HIPK3

Ling Zhou1, Ying Xiao2,(), Qiushi Li1, Zhaoyi Chen1, Qi Li3, Yuanming Wu1   

  1. 1. Department of Respiratory and Critical Care Medicine Wuhan Pu Ren Hospital Official Website,Wuhan 430080,China
    2. Department of hematology,Wuhan Pu Ren Hospital Official Website,Wuhan 430080,China
    3. Department of Internal Medicine,The fourth Clinical College of Wuhan University of Science and Technology,Wuhan 430080,China
  • Received:2025-03-25 Published:2025-06-25
  • Corresponding author: Ying Xiao
引用本文:

周玲, 肖颖, 李秋诗, 陈兆毅, 李琪, 吴园明. 亚麻木酚素通过circRNA HIPK3影响非小细胞肺癌A549 细胞凋亡及铁死亡的机制研究[J/OL]. 中华肺部疾病杂志(电子版), 2025, 18(03): 362-368.

Ling Zhou, Ying Xiao, Qiushi Li, Zhaoyi Chen, Qi Li, Yuanming Wu. Mechanism of secoisolariciresinol diglucoside influencing apoptosis and ferroptosis in non-small cell lung cancer A549 cells through circRNA HIPK3[J/OL]. Chinese Journal of Lung Diseases(Electronic Edition), 2025, 18(03): 362-368.

目的

分析亚麻木酚素(secoisolariciresinol diglucoside,SDG)通过环状RNA 同源域相互作用蛋白激酶3(circular RNA homeodomain interacting protein kinase 3,circRNA HIPK3)影响非小细胞肺癌(non-small cell lung cancer,NSCLC)A549 细胞凋亡及铁死亡的机制。

方法

肺癌A549 细胞分为A549组、SDG-L 组、SDG-M 组、SDG-H 组、SDG-H+oe-NC 组、SDG-H+oe-circRNA HIPK3 组。 每组实验重复6 次。 噻唑蓝实验检测细胞存活率;荧光定量PCR 实验检测circRNA HIPK3、NADPH 氧化酶1(nicotinamide adenine dinucleotide phosphate oxidase 1,NOX1)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、铁蛋白重链1(ferritin heavy chain 1,FTH1)、溶质载体家族7 成员11(solute carrier family 7 member 11,SLC7A11)的mRNA 表达;试剂盒检测Fe2+、丙二醛(malondialdehyde,MDA)、谷胱甘肽(glutathione,GSH)水平;2',7'-二氯荧光素二乙醇酯染色法检测活性氧(reactive oxygen species,ROS)水平;蛋白免疫印记法测定NOX1、GPX4、FTH1、SLC7A11、核因子E2 相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)和血红素氧合酶1(heme oxygenase-1,HO-1)的表达变化;Transwell 和原位末端标记实验检测细胞侵袭和凋亡。

结果

不同浓度SDG 对人支气管上皮细胞HBE1 的存活率无显著影响;降低A549 细胞存活率,SDG-H 组细胞存活率(55.29±10.05)%降低最显著,选择40 μmol/L SDG 进行后续实验。 circRNA HIPK3 在A549 组(2.36±0.33)细胞中高表达,SDG-H 组(1.72±0.26)细胞中低表达。SDG-H 组细胞Fe2+(4.98±0.93)mmol/L、MDA(5.72±1.26)μmol/g prot、ROS(2.12±0.81)、细胞凋亡率(29.74±10.05)% 高于A549 组(3.25±0.49)mmol/L、(3.36±0.72)μmol/g prot、(1.00±0.19)、(13.84±6.17)%(P<0.01),NOX1 的mRNA 和蛋白表达高于A549 组(P<0.01),GSH(3.94±0.63)μmol/g prot、细胞侵袭数(45.92±16.88)个低于A549 组(6.08±1.71)μmol/g prot、(117.33±37.14)个(P<0.05),GPX4、FTH1、SLC7A11 的mRNA 和蛋白表达、Nrf2 和HO-1 蛋白水平低于A549 组(P <0.05)。 SDG-H+oecircRNA HIPK3 组细胞Fe2+(4.13±0.41)mmol/L、MDA(3.88±0.94)μmol/g prot、ROS(1.49±0.42)、细胞凋亡率(19.16±8.30)%低于SDG-H+oe-NC 组(5.11±0.76)mmol/L、(5.65±1.11)μmol/g prot、(2.26±0.67)、(33.21±11.72)%(P<0.05),NOX1 的mRNA 和蛋白表达低于SDG-H+oe-NC 组(P<0.05),GSH(5.87±1.45)μmol/g prot、细胞侵袭数(92.24±22.72)个高于SDG-H+oe-NC 组(4.11±0.70)μmol/g prot、(54.41±19.10)个(P<0.05),GPX4、FTH1、SLC7A11 的mRNA 和蛋白表达、Nrf2 和HO-1 蛋白水平高于SDG-H+oe-NC 组(P<0.05)。

结论

SDG 通过调控circRNA HIPK3,促进NSCLC A549 细胞凋亡,抑制细胞侵袭可能与抑制Nrf2/HO-1 抗氧化通路激活细胞铁死亡具有意义。

Objective

To analyze the mechanism of secoisolariciresinol diglucoside (SDG) affecting apoptosis and ferroptosis of non-small cell lung cancer A549 cells through circular RNA homeodomain interacting protein kinase 3 (circRNA HIPK3).

Methods

Lung cancer A549 cells were divided into A549 group,SDG-L group,SDG-M group,SDG-H group,SDG-H+oe-NC group,SDG-H+oe-circRNA HIPK3 group.Each experiment was repeated 6 times. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to detect the cell viability. The mRNA expressions of circRNA HIPK3,nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1),glutathione peroxidase 4 (GPX4),ferritin heavy chain 1 (FTH1),and solute carrier family 7 member 11 (SLC7A11). Kits were used to detect the levels of Fe2+,malondialdehyde (MDA),and glutathione (GSH). The 2',7'-dichlorofluorescin diacetate staining method was used to detect the level of reactive oxygen species (ROS). Western blot was used to determine the expression changes of NOX1,GPX4,FTH1,SLC7A11,nuclear factor erythroid 2-related factor 2 (Nrf2),and heme oxygenase-1 (HO-1). The Transwell assay and terminal deoxynucleotidyl transferase dUTP nick end labeling assay were used to detect cell invasion and apoptosis.

Results

SDG at different concentrations had no significant effect on the survival rate of human bronchial epithelial cells HBE1,but it decreased the survival rate of A549 cells. The cell viability in the SDG-H group [(55.29±10.05) %] decreased most significantly,and 40 μmol/L of SDG was selected for subsequent experiments. Circular RNA HIPK3 was highly expressed in the cells of the A549 group (2.36±0.33) and lowly expressed in the cells of the SDG-H group (1.72±0.26).The levels of Fe2+,MDA,ROS and the cell apoptosis rate in the SDG-H group were higher than those in the A549 group (P<0.01). The mRNA and protein expressions of NOX1 were higher than those in the A549 group(P<0.01). The levels of GSH and the number of cell invasions (P<0.05). The mRNA and protein expressions of GPX4,FTH1,SLC7A11,and the protein levels of Nrf2 and HO-1 were lower than those in the A549 group(P<0.05). The levels of Fe2+,MDA,ROS and the cell apoptosis rate in the cells of the SDG-H+oe-circRNA HIPK3 group were lower than those in the SDG-H+oe-NC group (P<0.05). The mRNA and protein expressions of NOX1 were lower than those in the SDG-H+oe-NC group (P<0.05). The levels of GSH and the number of cell invasions were higher than those in the SDG-H+oe-NC group(P<0.05). The mRNA and protein expressions of GPX4,FTH1,SLC7A11,and the protein levels of Nrf2 and HO-1 were higher than those in the SDG-H+oe-NC group (P <0.05).

Conclusion

SDG promoted apoptosis of non-small cell lung cancer A549 cells and inhibited cell invasion by regulating circRNA HIPK3. This might be related to inhibiting the activation of the Nrf2/HO-1 antioxidant pathway and activating ferroptosis in cells.

表1 每组中Fe2+、MDA、GSH 水平结果(
表2 每组中铁死亡相关标志物mRNA 相对表达水平结果(
表3 每组中铁死亡相关标志物蛋白相对表达水平结果(
表4 每组细胞侵袭个数和凋亡率比较(
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