缺氧条件下HIF-1α/ROS对肺癌A549细胞凋亡和侵袭的作用及其机制

doi:10.13481/j.1671-587X.20230317

摘要/Abstract

摘要：

目的探讨缺氧条件下缺氧诱导因子1α（HIF-1α）/活性氧（ROS）对非小细胞肺癌A549细胞凋亡和侵袭的作用，并阐明其相关机制。方法将A549细胞置于缺氧条件下培养12、24、48和72 h，构建缺氧细胞模型，实时荧光定量PCR（RT-qPCR）法检测不同处理时间细胞中HIF-1α mRNA表达水平，CCK-8法检测细胞增殖能力，鉴定缺氧细胞模型并确定最佳诱导时间。将A549细胞分为对照组（21%O₂）、模型组（缺氧诱导）、N-乙酰半胱氨酸（NAC）组（20 mmol·L^－1 NAC）、NAC+利非西呱（YC-1）组（20 mmol·L^－1NAC+100 μmol·L^－1 YC-1）和YC-1组（100 μmol·L^－1 YC-1）。RT-qPCR和Western blotting法检测各组细胞中HIF-1α和人甲酰肽受体1（FPR1） mRNA及蛋白表达水平，流式细胞术检测各组细胞中ROS水平，透射电镜观察各组细胞自噬体结构，免疫荧光法检测各组细胞中微管相关轻链3Ⅱ（LC3-Ⅱ）表达情况，流式细胞术检测各组细胞凋亡率，Transwell小室实验检测各组侵袭细胞数。结果随着低氧诱导时间的延长，A549细胞中HIF-1α mRNA表达水平和细胞增殖能力均明显升高（P<0.05或P<0.01），最佳低氧诱导时间为24 h。RT-qPCR和Western blotting法检测，与对照组比较，模型组细胞中HIF-1α和FPR1 mRNA及蛋白表达水平明显升高（P<0.01）；与模型组比较，NAC组、NAC+YC-1组和YC-1组细胞中HIF-1α和FPR1 mRNA及蛋白表达水平均明显降低（P<0.01）；与NAC组比较，NAC+YC-1组细胞中HIF-1α和FPR1 mRNA及蛋白表达水平明显降低（P<0.01）。流式细胞术检测，与对照组比较，模型组细胞中ROS水平明显升高（P<0.01）；与模型组比较，NAC组、NAC+YC-1组和YC-1组细胞中ROS水平均明显降低（P<0.01）；与NAC组比较，NAC+YC-1组细胞中ROS水平明显降低（P<0.01）。透射电镜观察，对照组细胞中细胞器结构完整，未见自噬体结构；模型组细胞中可见大量自噬体和明显空泡，细胞中细胞器被降解；与模型组比较，NAC组、NAC+YC-1组和YC-1组细胞中自噬体减少，其中NAC+YC-1组细胞中自噬体最少。免疫荧光法检测，与对照组比较，模型组细胞中LC3-Ⅱ表达强度明显增加；与模型组比较，NAC组、NAC+YC-1组和YC-1组细胞中LC3-Ⅱ表达强度明显减少。流式细胞术检测，与对照组比较，模型组细胞凋亡率明显降低（P<0.01）；与模型组比较，NAC组、NAC+YC-1组和YC-1组细胞凋亡率明显升高（P<0.01）；与NAC组比较，NAC+YC-1细胞凋亡率明显升高（P<0.01）。Transwell小室实验检测，与对照组比较，模型组侵袭细胞数明显增加（P<0.01）；与模型组比较，NAC组、NAC+YC-1组和YC-1组侵袭细胞数均明显减少（P<0.01）；与NAC组比较，NAC+YC-1组侵袭细胞数明显减少（P<0.01）。结论缺氧条件下抑制HIF-1α/ROS可促进肿瘤细胞凋亡，并抑制细胞侵袭，其作用机制与抑制肺癌细胞自噬有关。

关键词: 缺氧, 缺氧诱导因子1α, 活性氧, 自噬, 癌，非小细胞肺

Abstract:

Objective To discuss the effect of hypoxia inducible factor-1α （HIF-1α）/ reactive oxygen species （ROS） on the apoptosis and invasion of the non-small cell lung cancer A549 cells，and to clarify the related mechanism. Methods The A549 cells were cultured under hypoxia condition for 12， 24， 48 and 72 h to construct the hypoxia cell model. The expression levels of HIF-1α mRNA in the cells at different treatment time were detected by real-time fluorescence quantitative PCR（RT-qPCR） method， and the proliferation abilities of the cells were detected by CCK-8 assay；the hypoxia cell model was verified， and the optimal induction time was determined.The A549 cells were divided into control group（21%O₂）， model group（hypoxia induction）， NAC group（20 mmol·L^－1 NAC）， NAC+YC-1 group（20 mmol·L^－1 NAC+100 μmol·L^－1 YC-1） and YC-1 （100 μmol·L^－1 YC-1）group. The expression levels of HIF-1α and FPR1 mRNA and proteins were detected by RT-qPCR and Western blotting methods； the ROS levels were detected by flow cytometry； the autophagosome structure in the cells was observed by transmission electron microscope； the expressions of microtubule-associated protein light china 3 Ⅱ（LC3-Ⅱ） in the cells in various groups were detected by immunofluorescence； the apoptotic rates of the cells in various groups were detected by flow cytometry； and the number of invasion cells was detected by Transwell chamber assay. Results With the prolongation of hypoxia induction time， the HIF-1α mRNA expression levels in the A549 cells and cell proliferation abilities were increased（P<0.05 or P<0.01）， and the optimal hypoxia induction time was 24 h. The results of RT-qPCR and Western blotting methods showed that compared with control group，the expression levels of HIF-1α and FPR1 mRNA and proteins in the cells in model group were significantly increased（P<0.01）； compared with model group，the expression levels of HIF-1α and FPR1 mRNA and proteins in the cells in NAC group， NAC+YC-1 group and YC-1 group were significantly decreased（P<0.01）.Compared with control group，the level of ROS in the cells in model group was significantly increased （P<0.01）； compared with model group，the ROS levels in the cells in NAC group， NAC+YC-1 group， and YC-1 group were significantly decreased（P<0.01）；compared with NAC group，the ROS level in the cells in NAC+YC-1 group was significantly decreased（P<0.01）.The transmission electron microscope results showed that the organelle structure of the cells in control group was intact and no autophagosome structure was seen； a large number of autophagosomes were seen in the cells in model group， obvious vacuoles were visible， and intracellular organelles were degraded； compared with model group， the number of autophagosomes in the cells in NAC group， NAC+YC-1 group and YC-1 group were decreased，among them the autophagosomes in the cells in NAC+YC-1 group were the least. The immunofluorescence assay results showed that the expression intensity of LC3-Ⅱ in the cells in model group was significantly increased compared with control group， and the expression intensities of LC3-Ⅱ in the cells in NAC group， NAC+YC-1 and YC-1 group were signficantly decreased compared with model group.Compared with control group， the apoptotic rate of the cells in model group was significantly decreased（P<0.01）； compared with model group， the apoptotic rates of the cells in NAC group， NAC+YC-1 group and YC-1 group were significantly increased（P<0.01）； compared with NAC group，the apoptotic rate of the cells in NAC+YC-1 group was increased（P<0.01）.The results of Transwell chamber assay showed that compared with control group the number of the invasion cells in model group was significantly increased（P<0.01）； compared with model group， the numbers of invasion cells in NAC group， NAC+YC-1 group and YC-1 group were significantly decreased （P<0.01）；compared with NAC group，the number of the in vasion cells in NAC+YC-1 group was decreased（P<0.01）. Conclusion Inhibition of HIF-1α/ROS under hypoxia condition can promote the apoptosis and inhibit cell invasion of cancer cells，and its mechanism may be realted to inhibiting the autophagy of lung cancer cells.

Key words: Hypoxia, Hypoxia inducible factor-1α, Reactive oxygen species, Autophagy, Cancer，non-small cell lung

中图分类号:

R734.2

黄波,丁洁,郭红荣,王红娟,徐建群,郑泉. 缺氧条件下HIF-1α/ROS对肺癌A549细胞凋亡和侵袭的作用及其机制[J]. 吉林大学学报(医学版), 2023, 49(3): 682-690.

Bo HUANG,Jie DING,Hongrong GUO,Hongjuan WANG,Jianqun XU,Quan ZHENG. Effects of HIF-1α/ROS on apoptosis and invasion of lung cancer A549 cells under hypoxia and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2023, 49(3): 682-690.

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参考文献 23

1	SCHABATH M B， COTE M L. Cancer progress and priorities： lung cancer［J］. Cancer Epidemiol Biomarkers Prev， 2019， 28（10）： 1563-1579.
2	ALEXANDER M， KIM S Y， CHENG H Y. Update 2020： management of non-small cell lung cancer［J］. Lung， 2020， 198（6）： 897-907.
3	WU F Y， WANG L， ZHOU C C. Lung cancer in China： current and prospect［J］. Curr Opin Oncol， 2021， 33（1）： 40-46.
4	DUMA N， SANTANA-DAVILA R， MOLINA J R. Non-small cell lung cancer： epidemiology， screening， diagnosis， and treatment［J］. Mayo Clin Proc， 2019， 94（8）： 1623-1640.
5	HUA Q， MI B M， XU F，et al.Hypoxia-induced lncRNA-AC020978 promotes proliferation and glycolytic metabolism of non-small cell lung cancer by regulating PKM2/HIF-1α axis［J］.Theranostics，2020，10（11）：4762-4778.
6	AJDUKOVIĆ J. HIF-1： a big chapter in the cancer tale［J］. Exp Oncol， 2016， 38（1）： 9-12.
7	ONORATI A V， DYCZYNSKI M， OJHA R， et al. Targeting autophagy in cancer［J］.Cancer，2018，124（16）： 3307-3318.
8	SHIDA M， KITAJIMA Y， NAKAMURA J， et al. Impaired mitophagy activates mtROS/HIF-1α interplay and increases cancer aggressiveness in gastric cancer cells under hypoxia［J］. Int J Oncol， 2016， 48（4）：1379-1390.
9	JEONG J K， GURUNATHAN S， KANG M H， et al. Hypoxia-mediated autophagic flux inhibits silver nanoparticle-triggered apoptosis in human lung cancer cells［J］. Sci Rep， 2016， 6： 21688.
10	YU H L， CHEN B， REN Q. Baicalin relieves hypoxia-aroused H9c2 cell apoptosis by activating Nrf2/HO-1-mediated HIF1α/BNIP3 pathway［J］. Artif Cells Nanomed Biotechnol， 2019， 47（1）： 3657-3663.
11	MAO X W， NANZHANG N， XIAO J， et al. Hypoxia-induced autophagy enhances cisplatin resistance in human bladder cancer cells by targeting hypoxia-inducible factor-1α［J］. J Immunol Res， 2021， 2021： 8887437.
12	TIRPE A A， GULEI D， CIORTEA S M， et al. Hypoxia： overview on hypoxia-mediated mechanisms with a focus on the role of HIF genes［J］. Int J Mol Sci， 2019， 20（24）： 6140.
13	CUI W， WU F， MA L. Hypoxia associated biomarkers in lung cancer - an update［J］. Eur Rev Med Pharmacol Sci， 2017， 21（3 ）： 43-46.
14	LI Y N， ZHANG Y S， LI X R， et al. Status of hypoxia-inducible factor-1α expression in non-small cell lung cancer［J］. Pharmazie， 2021， 76（9）： 404-411.
15	MOLONEY J N， COTTER T G. ROS signalling in the biology of cancer［J］.Semin Cell Dev Biol，2018，80：50-64.
16	黄波，郭红荣，丁洁，等. 肺腺癌中FPR1的表达及其对细胞迁移、成瘤能力的影响［J］. 临床与实验病理学杂志， 2018， 34（10）： 1104-1109.
17	LI X H， HE S K， MA B Y. Autophagy and autophagy-related proteins in cancer［J］.Mol Cancer，2020，19（1）：12.
18	ARIOSA A R， LAHIRI V， LEI Y C，et al. A perspective on the role of autophagy in cancer［J］. Biochim Biophys Acta Mol Basis Dis， 2021， 1867（12）： 166262.
19	TUREK K， JAROCKI M， KULBACKA J， et al. Dualistic role of autophagy in cancer progression［J］. Adv Clin Exp Med， 2021， 30（12）： 1303-1314.
20	DENG M Z， ZHANG W Z， YUAN L L， et al. HIF-1a regulates hypoxia-induced autophagy via translocation of ANKRD37 in colon cancer［J］. Exp Cell Res， 2020， 395（1）： 112175.
21	JOSHI S， KUMAR S， PONNUSAMY M P， et al. Hypoxia-induced oxidative stress promotes MUC4 degradation via autophagy to enhance pancreatic cancer cells survival［J］. Oncogene， 2016， 35（45）： 5882-5892.
22	TANIDA I， UENO T， KOMINAMI E. LC3 and autophagy［J］. Methods Mol Biol， 2008， 445： 77-88.
23	FAN S J， YUE L Y， WAN W， et al. Inhibition of autophagy by a small molecule through covalent modification of the LC3 protein［J］. Angew Chem Int Ed Engl， 2021， 60（50）： 26105-26114.

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