SLC2A1抑制肺腺癌铁死亡并促进肺腺癌细胞的增殖和侵袭

doi:10.12122/j.issn.1673-4254.2024.12.17

摘要/Abstract

摘要：

目的探讨细胞能量代谢中的葡萄糖转运蛋白SLC2A1对肺腺癌增殖和迁移能力的影响及其分子机制。方法分析TCGA数据库中SLC2A1基因在肺腺癌正常组织和肿瘤组织中的表达差异及其对预后的影响。收集我院临床肺腺癌患者的肿瘤组织和癌旁配对组织，进行免疫组化染色，分析SLC2A1蛋白在肿瘤中的表达水平。验证肺腺癌肿瘤组织和癌旁组织中SLC2A1蛋白的表达差异，探讨该蛋白与患者临床病理特征之间的关系。体外构建稳定过表达和稳定干预SLC2A1基因的细胞系，通过Western blotting实验和qRT-PCR实验验证干预效果。采用CCK-8和Transwell实验体外验证细胞表型，明确其对肿瘤细胞增殖、迁移能力的影响。检测干预SLC2A1后细胞的铁死亡和细胞自噬相关蛋白的表达水平，并通过ROS荧光染色、Fe²⁺荧光染色明确铁死亡的发生过程。结果与正常肺组织相比，SLC2A1在肺腺癌肿瘤组织中高表达（P<0.05），且与肺腺癌患者的病理参数及不良预后相关（P<0.05）。与对照组相比，过表达SLC2A1，细胞中该基因上调（P<0.05），细胞增殖活性增加（P<0.05），细胞侵袭和迁移能力增强（P<0.05）。干预SLC2A1基因表达后，细胞死亡比例增加（P<0.05），侵袭和增殖能力均被抑制（P<0.05）。干预SLC2A1激活了铁死亡，GPX4、xCT等铁死亡指标表达下调，细胞内ROS累积，Fe²⁺增多（P<0.05）。同时检测到细胞自噬的发生，LC3B增加，且这一过程可被3-MA挽救。结论 SLC2A1在肿瘤组织中高表达且与不良预后有关。靶向抑制SLC2A1可以促进肺腺癌中的铁死亡和自噬发生，有望成为肺腺癌诊断和治疗的新潜在分子靶点。

关键词: 肺腺癌, 葡萄糖转运蛋白1, 铁死亡, 细胞自噬

Abstract:

Objective To examine how the glucose transporter SLC2A1 influences the proliferation and migration of lung adenocarcinoma (LUAD) and explore the underlying molecular mechanisms. Methods We examined the differential expression of SLC2A1 between normal and LUAD tissues in the TCGA database and its prognostic implications. Immunohistochemistry was used to detect SLC2A1 protein levels in clinical samples of LUAD and adjacent tissues, and the association of SLC2A1 expression levels with clinicopathological features of the patients was analyzed. In PC9 cells with stable SLC2A1 overexpression or knockdown, the effects of SLC2A1 expression level on cell proliferation and migration were assessed using CCK-8 and Transwell assays, and the changes in expressions of ferroptosis- and autophagy-related proteins were measured; the occurrence of ferroptosis was confirmed using ROS and Fe²⁺ fluorescence staining. Results SLC2A1 expression was significantly higher in LUAD tumor tissues than in normal lung tissues (P<0.05) and was associated with worse pathological parameters and prognosis of the patients (P<0.05). In PC9 cells, SLC2A1 overexpression significantly promoted cell proliferation, invasion and migration, and SLC2A1 knockdown significanty increased cell death and inhibited cell invasion and proliferation. SLC2A1 knockdown caused obvious activation of cell ferroptosis, reduced GPX4 and xCT expressions, and increased intracellular levels of ROS and Fe²⁺. SLC2A1 knockdown also resulted in increased cell autophagy shown by increased LC3B expression, which could be reversed by treatement with 3-MA. Conclusion High SLC2A1 expression is correlated with poor prognosis of patients with LUAD, and inhibiting SLC2A1 can induce ferroptosis and autophagy of LUAD cells, suggesting the potential of SLC2A1 as a target for LUAD diagnosis and treatment.

Key words: lung adenocarcinoma, glucose transporter 1, ferroptosis, autophagy

匡红, 蔡文涵, 刘一鸣, 温佳新, 田硕, 薛志强. SLC2A1抑制肺腺癌铁死亡并促进肺腺癌细胞的增殖和侵袭[J]. 南方医科大学学报, 2024, 44(12): 2404-2411.

Hong KUANG, Wenhan CAI, Yiming LIU, Jiaxin WEN, Shuo TIAN, Zhiqiang XUE. High expression of SLC2A1 inhibits ferroptosis and promotes proliferation and invasion of lung adenocarcinoma cells[J]. Journal of Southern Medical University, 2024, 44(12): 2404-2411.

图/表 9

图1 SLC2A1在肺腺癌和正常组织表达情况

Fig.1 Expression of SLC2A1 in lung adenocarcinoma (LUAD) and normal lung tissues and cells. A: Expression of SLC2A1 in LUAD and normal lung tissue. B: Expression of SLC2A1 in LUAD patients with different T grades. C: Expression of SLC2A1 in PC9 and BEAS-2B cell lines. *P<0.05, ****P<0.0001.

表1 肺腺癌患者临床特征与SLC2A1表达相关性

Tab.1 Relation between SLC2A1 and clinicopathological features of patients with LUAD (n)

Clinicopathological feature	n	SLC2A1 Low	SLC2A1 High	P
Gender				0.0577
Male	238	79	159
Female	278	112	166
Age (year)				<0.0001
<65	365	135	230
≥65	151	67	84
Tumor diameter (cm )				0.0351
<4	280	136	144
≥4	236	62	174
Fuhrman				0.2935
1-2	353	155	198
3-4	163	58	105

图2 SLC2A1在肺腺癌诊断和预后情况

Fig.2 Diagnostic and prognostic values of SLC2A1 in LUAD. A: AUC curve feature of SLC2A1 in LUAD. B: Overall survival of LUAD patients with different levels of SLC2A1. C: Progression-free survival of LUAD patients with different levels of SLC2A1.

表2 单因素分析SLC2A1表达与肺腺癌患者临床特征及预后相关性

Tab.2 Univariate analysis of the correlations of SLC2A1 expression and clinicopathologic variables with overall survival of LUAD patients

Variable	n	Mean Survival (months)	P
Gender			0.2986
Male	156	69.2
Female	51	62.3
Age (year)			0.0620
<65	165	70.0
≥65	42	63.6
Tumor diameter ( cm )			0.0053
<4	71	71.2
≥4	136	67.0
Fuhrman			0.0270
1-2	153	71.7
3-4	54	60.1
SLC2A1 expression			0.0079
Low	59	71.7
High	148	69.2
T stage			0.1082
T1	52	71.0
T2	77	70.4
T3	78	65.5

表3 COX多因素分析临床指标相关性

Tab.3 COX multivariate analysis of clinicopathological parameters and overall survival of the patients

Variables	Hazard Ratio (95% CI)	P
Tumor diameter	1.239(1.136-1.352)	0.220
SLC2A1 expression	1.912(1.841-1.988)	0.024
Fuhrman	2.029(1.995-2.063)	0.194

图3 SLC2A1蛋白在肺腺癌与正常组织中的表达情况

Fig.3 Expression of SLC2A1 protein in LUAD and normal lung tissues. ****P<0.0001.

图4 干预SLC2A1抑制肺腺癌细胞的增殖迁移能力

Fig.4 Knockdown of SLC2A1 inhibits proliferation and migration of LUAD cells. A: Efficiency of SLC2A1 knockdown in PC9 cells. B: qRT-PCR data showing SLC2A1 mRNA expression in PC9 with or without shRNA. C: CCK-8 assay for assessing proliferation of PC9 cells with SLC2A1 downregulation. D: Migration ability of PC9 cells with SLC2A1 downregulation. E: Invasion ability of PC9 cells with SLC2A1 downregulation. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

图5 过表达SLC2A1促进肺腺癌细胞的增殖迁移能力

Fig.5 Overexpression of SLC2A1 promotes proliferation and migration of LUAD cells.A: Efficiency of SLC2A1 overexpression in PC9 cells. B: qRT-PCR of SLC2A1 mRNA expression in PC9 cells with or without SLC2A1 overexpression. C: CCK-8 for assessing proliferation of PC9 cells overexpressing SLC2A1. D: Migration ability of PC9 cells overexpressing SLC2A1. E: Invasion of PC9 cells overexpressing SLC2A1. Scar bar=100 μm. **P<0.01, ***P<0.001, ****P<0.0001 vs control group.

图6 干预SLC2A1促进肺腺癌发生自噬与铁死亡

Fig.6 SLC2A1 knockdown promotes autophagy and iron death in PC9 cells. A: Western blotting of ferroptosis-related proteins in PC9 cells with SLC2A1 knockdown. B: Intracellular Fe2+ level in PC9 cells with SLC2A1 knockdown. C: Mito-Fe2+ level in PC9 cells with SLC2A1 knockdown. D: Intracellular ROS level in PC9 cells with SLC2A1 knockdown. E: Knockdown of SLC2A1 induces autophagy in PC9 cells. F: 3-MA resuced SLC2A1 knockdown-induced autophagy of PC9 cells. G: Knockdown of SLC2A1 induces cell death in PC9 cells. Scar bar=100 μm. **P<0.01, ***P<0.001, ****P<0.0001.

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