| Background: Lung cancer is one of the malignant tumors with high incidence and mortality.Among all primary lung cancers,non-small cell lung cancer(NSCLC)is the most commonly diagnosed pathological type,accounting for about 85%-90%.NSCLC is prone to metastasis and most patients are at middle and advanced stages at the time of diagnosis,when surgery is inoperable.Despite great advances in treatment modalities such as targeted therapy and immunotherapy in the past few decades,the prognosis of patients with NSCLC remains poor,mainly due to the existence of cancer stem cells(CSCs).CSCs are a small subgroup of cancer cells which have the potential of self-renewal,extensive replication and multiline differentiation.They are the root cause of recurrence,metastasis and drug resistance of NSCLC.Targeting cancer stem cells is an effective way to improve patient clinical outcomes.Abnormal iron metabolism can lead to the occurrence and development of tumors.Increased iron accumulation and iron dependence are one of the characteristics of cancer stem cells.Specifically targeting iron metabolism in cancer stem cells can improve the efficacy of tumor therapy.Ferroptosis is a non-apoptotic cell death process driven by ironregulated lipid peroxidation.Tumor cells,especially cancer stem cells resistant to multidrug therapy,can be specifically targeted by ferroptosis activators due to their high ROS load and iron dependence to achieve excellent antitumor effects.Studies have confirmed that targeting the ferroptosis pathway can be used as a potential strategy for the treatment of non-small cell lung cancer.Therefore,finding the key pathways that induce ferroptosis may provide new insights into the treatment of NSCLC.The translation process is a key step in the regulation of stem cell fate.Eukaryotic translation initiation factor 3a(eIF3a),the largest subunit of eIF3,has been demonstrated to play an important role in the development of a variety of tumors.eIF3 a plays a key role in development,metastasis and chemotherapy resistance of NSCLC,but hitherto whether eIF3 a is involved in the maintenance of NSCLC stem cell-like characteristics remains unclear.In addition,previous studies have shown that stress stimulation caused by iron downregulation can inhibit the expression of eIF3 a,but whether eIF3 a can regulate the iron-dependent ferroptosis in a feedback way has not been reported yet.Further in-depth investigation of the effects and molecular mechanisms of eIF3 a on regulating NSCLC stem-like traits and ferroptosis,can supplement our understanding of the biological function of eIF3 a,and also provide theoretical basis and guidance for targeting eIF3 a as a promising therapeutic strategy for NSCLC.Objectives: To investigate the roles and molecular mechanisms of eIF3 a in maintaining stem-like characteristics and regulating ferroptosis in NSCLC.Methods: Gene Set Enrichment Analysis(GSEA)was performed to explore the association between eIF3 a expression and stem-cell related gene signaling pathways by using the TCGA lung adenocarcinoma dataset,and the association was validated in in-house lung cancer tissues through quantitative real-time PCR(RT-PCR)assay in collected lung cancer tissues.The effects of eIF3 a on the stem cell-like characteristics of NSCLC were investigated by sphere formation,colony formation,flow cytometry and western blot assays after knockdown of eIF3 a.The regulation of eIF3 a on NSCLC cell metastasis was detected by wound healing and transwell assays.A subcutaneous xenograft tumor model was established and the limiting dilution assay was used to investigate whether eIF3 a affect the tumorigenic ability of NSCLC cells.Bone marrow mesenchymal stem cells extracted from eIF3 a knockout mice were cultured in vitro to observe the effects of eIF3 a on the growth rate and morphology of normal stem cells.The regulatory mechanisms of eIF3 a on Wnt/β-catenin pathway were explored through western blot,RT-PCR,immunohistochemistry,dualluciferase reporter,co-immunoprecipitation,immunofluorescence,RNAimmunoprecipitation(RIP)assays.The downstream proteins regulated by eIF3 a were identified by proteomic analysis,and the transcription factor of interest were verified by western blot,RT-PCR,and dual-luciferase reporter gene assays.KEGG enrichment analysis of the proteomic data was performed to explore biological processes in which eIF3 a is significantly involved.The expression levels of ferroptosis-related genes in the proteomic data were compared between the control group and eIF3 a knockdown group.CCK-8,plate colony,and Calcein-AM/PI staining were used to determine whether intervention of eIF3 a expression affects RSL3-induced ferroptosis.Detecting the levels of Lipid-ROS and MDA by flow cytometry to investigate whether the expression of eIF3 a affects the lipid peroxidation induced by RSL3.Mitochondrial membrane potential after eIF3 a knockdown was detected by JC-1 probe.The effect of eIF3 a knockdown on regulating RSL3-induced polyunsaturated fatty acid phospholipids(PUFA-PLs)levels was explored by lipid omics analysis.Results:(1)Through GSEA enrichment analysis,we found that eIF3 a was significantly positively correlated with the activation of stem cellrelated signaling pathways;RT-PCR showed that the expression of eIF3 a in NSCLC tissues was positively correlated with the expression of the stem cell markers CD133 and CD44;In addition,compared with adherent cells,the expression of eIF3 a was significantly up-regulated in the sphere cells.(2)After knockdown of eIF3 a expression,the ability of NSCLC to form spheres and colonies was significantly reduced;Flow cytometry results showed that eIF3 a knockdown inhibited the cell surface expression of CD44,a marker of lung cancer stem cells;Western blot results indicated that eIF3 a could significantly affect the protein expression of SOX2,OCT4 and NANOG.(3)According to the subcutaneous tumor xenograft model by using limiting dilution assay,eIF3 a could affect the tumorigenic ability of NSCLC cells at different cell concentrations.In addition,eIF3 a knockdown could affect the ability of NSCLC to metastasize in vitro and also inhibit the growth of normal stem cells.(4)By GSEA enrichment analysis,we found that high expression of eIF3 a was significantly positively correlated with the activity of Wnt/β-catenin signaling pathway in NSCLC;co-IP results confirmed that eIF3 a could affect the binding ofβ-catenin to TCF4;Dual-luciferase reporter assay showed that eIF3 a could regulate β-catenin-TCF4 transcription complex-mediated transcription of downstream target genes;eIF3 a could regulate the transcription and protein expression of downstream target genes of Wnt/β-catenin;A series of rescue experiments demonstrated that Wnt/β-catenin pathway is responsible for mediating eIF3 a regulation of NSCLC stem cell-like properties.(5)The actinomycin tracing experiment showed that eIF3 a had no significant effect on the stability of β-catenin protein;The immunofluorescence,and nuclear and cytoplasmic protein detection showed that eIF3 a could affect the expression of β-catenin protein in the nucleus;RIP experiment showed that the m RNA binding of eIF3 a and β-catenin was not significant.RT-PCR and dual-luciferase reporter gene assay confirmed that eIF3 a could regulate m RNA transcription of β-catenin.(6)The potential transcription factors regulating β-catenin transcription were predicted by the PROMO database;Protein spectrum analysis suggested that YY1 might mediate the transcription regulation of β-catenin by eIF3a;Dual-luciferase reporter assay and RT-PCR confirmed that eIF3 a regulated the transcription of β-catenin by YY1.(7)KEGG enrichment analysis of proteomics data found that eIF3 a was related to multiple signaling pathways and biological processes related to ferroptosis,and the expression of multiple driver genes and inhibitory genes of ferroptosis changed significantly with the downregulation of eIF3 a expression;CCK8 and Calcein-AM/PI staining experiments confirmed that down-regulation of eIF3 a can promote cell death induced by ferroptosis activator RSL3;flow cytometry detection of lipid-ROS and MDA levels found that silencing eIF3 a can enhance lipid peroxidation induced by RSL3;JC-1 probe detection proved that silencing eIF3 a can exacerbate RSL3-induced downregulation of mitochondrial membrane potential;lipidomics analysis showed that RSL3 can induce the upregulation of PUFA-PLs in NSCLC cells,and the PUFA-PLs induced by RSL3 upregulated more significantly after knockdown of eIF3 a.Conclusions:(1)Expression of eIF3 a is positively associated with stem-like characteristics of non-small cell lung cancer;eIF3 a knockdown significantly inhibits tumor stem-like features and tumor metastasis in NSCLC.(2)Mechanistically,eIF3 a promotes the transcriptional activation of β-catenin through YY1,thus leading to its increased accumulation in the nucleus.Entry of β-catenin into the nucleus contributes to the formation ofβ-catenin-TCF4 transcriptional coactivator complex and promotes the expression of downstream target genes,thus facilitating maintenance of the stem cell-like characteristics of NSCLC.However,eIF3 a had no significant effect on the translation and degradation of β-catenin.(3)eIF3 a is closely related to lipid metabolism.eIF3 a knockdown significantly influences the expression of multiple driver and suppressor genes of ferroptosis;knockdown of eIF3 a can significantly promote cell death induced by GPX4 inhibitor RSL3,increase lipid peroxidation and downregulate mitochondrial membrane potential;eIF3 a knockdown significantly promotes RSL3-induced upregulation of PUFA-PLs. |
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