Irisin affects the proliferation and migration of lung adenocarcinoma cells by regulating the EBF3/ALOX15 pathway

doi:10.11958/20242182

Abstract

Abstract:

Objective To investigate the effect of irisin regulating early B cytokine 3 (EBF3)/arachidonic acid-15-lipoxygenase (ALOX15) pathway on the proliferation and migration of lung adenocarcinoma cells. Methods A549 cells were assigned into the irisin solvent group, the irisin group, the sh-NC group, the EBF3 inhibitor (sh-EBF3) group, the irisin+sh-NC group and the irisin+sh-EBF3 group randomly. 5-bromo-2-deoxyuracil (EdU) staining and CCK-8 method were applied to detect cell proliferation. 5-Scratch experiment was applied to detect the scratch healing rate. 2', 7'-dichlorofluorescein diacetate (DCFH-DA) staining was applied to detect the level of reactive oxygen species (ROS) in cells. The reagent kit was used to detect glutathione (GSH), malondialdehyde (MDA) and ferrous ion (Fe²⁺) in cells. Transmission electron microscopy was applied to observe mitochondrial morphology in A549 cells. QRT-PCR was applied to detect mRNA levels of proliferating cell nuclear antigen (PCNA), matrix metalloproteinase 2 (MMP-2) and glutathione peroxidase 4 (GPX4) in A549 cells. Western blot assay was applied to detect EBF3 and ALOX15 proteins in cells. Results Compared with the irisin solvent group, the mitochondria of A549 cells in the irisin group showed ferroptosis characteristics, the positive rate of EdU, OD₄₅₀ value, scratch healing rate, GSH level, PCNA, MMP-2 and GPX4 mRNA levels decreased, and the ROS relative fluorescence intensity, MDA, Fe²⁺ level, and EBF3 and ALOX15 protein levels increased (P<0.05). Compared with the sh-NC group, the mitochondrial ferroptosis phenomenon of A549 cells was reduced in the sh-EBF3 group, the positive rate of EdU, OD₄₅₀ value, scratch healing rate, GSH level, PCNA, MMP-2 and GPX4 mRNA levels increased, and the ROS relative fluorescence intensity, MDA, Fe²⁺ levels and EBF3 and ALOX15 protein levels reduced (P<0.05). Sh-EBF3 reversed the effect of irisin on ferroptosis, proliferation and migration of A549 cells. Conclusion Irisin may induce ferroptosis in A549 cells and inhibit cell proliferation and migration by activating the EBF3/ALOX15 pathway.

Key words: adenocarcinoma of lung, cell proliferation, cell movement, ferroptosis, irisin, early B-cell factor 3/arachidonate-15-lipoxygenase pathway

CLC Number:

R734.2

Figures/Tables 9

References 23

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组别	EdU阳性率/%	OD₄₅₀值	划痕愈合率/%
鸢尾素溶剂组	28.96±1.43	0.83±0.07	35.62±1.83
鸢尾素组	13.25±0.67^a	0.32±0.03^a	16.77±0.79^a
sh-NC组	29.05±1.39	0.83±0.08	35.16±1.72
sh-EBF3组	36.65±1.78^b	0.99±0.06^b	43.39±2.01^b
鸢尾素+sh-NC组	12.99±0.61	0.30±0.02	16.67±0.81
鸢尾素+sh-EBF3组	20.58±0.97^c	0.67±0.05^c	25.59±1.23^c
F	369.847^＊＊	158.605^＊＊	329.832^＊＊

组别	EdU阳性率/%	OD₄₅₀值	划痕愈合率/%
鸢尾素溶剂组	28.96±1.43	0.83±0.07	35.62±1.83
鸢尾素组	13.25±0.67^a	0.32±0.03^a	16.77±0.79^a
sh-NC组	29.05±1.39	0.83±0.08	35.16±1.72
sh-EBF3组	36.65±1.78^b	0.99±0.06^b	43.39±2.01^b
鸢尾素+sh-NC组	12.99±0.61	0.30±0.02	16.67±0.81
鸢尾素+sh-EBF3组	20.58±0.97^c	0.67±0.05^c	25.59±1.23^c
F	369.847^＊＊	158.605^＊＊	329.832^＊＊

组别	ROS/相对荧光强度	MDA/ （nmol/mg）	GSH/ （nmol/mg）	Fe²⁺/ （nmol/mg）
鸢尾素溶剂组	1.00±0.01	6.25±0.32	1.86±0.14	0.47±0.05
鸢尾素组	3.65±0.17^a	17.73±0.85^a	0.65±0.05^a	2.16±0.12^a
sh-NC组	0.98±0.02	6.25±0.92	1.85±0.08	0.43±0.03
sh-EBF3组	0.39±0.03^b	4.16±0.21^b	2.39±0.11^b	0.21±0.01^b
鸢尾素+sh-NC组	3.71±0.18	17.82±0.83	0.62±0.06	2.20±0.13
鸢尾素+sh-EBF3组	1.62±0.07^c	10.51±0.52^c	1.34±0.51^c	1.46±0.12^c
F	1 102.695^＊＊	490.363^＊＊	60.399^＊＊	597.505^＊＊

组别	ROS/相对荧光强度	MDA/ （nmol/mg）	GSH/ （nmol/mg）	Fe²⁺/ （nmol/mg）
鸢尾素溶剂组	1.00±0.01	6.25±0.32	1.86±0.14	0.47±0.05
鸢尾素组	3.65±0.17^a	17.73±0.85^a	0.65±0.05^a	2.16±0.12^a
sh-NC组	0.98±0.02	6.25±0.92	1.85±0.08	0.43±0.03
sh-EBF3组	0.39±0.03^b	4.16±0.21^b	2.39±0.11^b	0.21±0.01^b
鸢尾素+sh-NC组	3.71±0.18	17.82±0.83	0.62±0.06	2.20±0.13
鸢尾素+sh-EBF3组	1.62±0.07^c	10.51±0.52^c	1.34±0.51^c	1.46±0.12^c
F	1 102.695^＊＊	490.363^＊＊	60.399^＊＊	597.505^＊＊

组别	PCNA mRNA	MMP-2 mRNA	GPX4 mRNA
鸢尾素溶剂组	1.00±0.00	1.00±0.00	1.00±0.00
鸢尾素组	0.46±0.03^a	0.36±0.03^a	0.23±0.02^a
sh-NC组	0.99±0.01	1.02±0.02	1.01±0.01
sh-EBF3组	1.58±0.12^b	1.93±0.18^b	2.16±0.17^b
鸢尾素+sh-NC组	0.44±0.03	0.38±0.03	0.25±0.02
鸢尾素+sh-EBF3组	0.87±0.07^c	0.65±0.05^c	0.79±0.06^c
F	300.906^＊＊	331.705^＊＊	537.399^＊＊