Study On ACSL1 Inducing Ferroptosis As A Potential Therapeutic Target For Clear Cell Renal Cell Carcinoma

BackgroundRenal cancer is one of the most common malignant tumors in the urinary system,with a noticeable increase in incidence and mortality rates in recent years.Clear cell renal cell carcinoma（ccRCC）is the most common subtype of renal cancer,and its prognosis still needs improvement.Therefore,exploring and validating new therapeutic targets for ccRCC and developing more effective treatment methods have become urgent needs.ccRCC is characterized by lipid metabolism imbalance and intracellular lipid accumulation,making lipid metabolic enzymes potential targets for ccRCC treatment.The long-chain acyl-coenzyme A synthetase（ACSL）family is a core enzyme class in lipid metabolism,catalyzing the esterification reaction between long-chain fatty acids and coenzyme A to form acyl-Co A,playing crucial roles in various biological processes such as fatty acid elongation,oxidation,phospholipid synthesis,and protein acylation.This family includes five members in mammals:ACSL1,ACSL3-6,and their activities are essential for the proliferation and progression of various cancer cells.Despite extensive attention to the role of ACSL1 in multiple cancers,there is still a lack of in-depth research on its role in ccRCC.ACSL1,as a critical lipid metabolic enzyme in tumor cells,has been found to regulate ferroptosis in certain cancers.Ferroptosis,as a nontraditional programmed cell death mode,is characterized by iron-dependent excessive lipid peroxidation-induced cell death.During ferroptosis,iron ions(Fe²⁺)catalyze the production of large amounts of lipid peroxides,damaging cell membranes and leading to cellular dysfunction and death.Previous studies have shown that the use of ferroptosis inducers can significantly increase the death rate of ccRCC cell lines.However,detailed regulatory mechanisms and pathways of ferroptosis in ccRCC are still lacking.This study aims to elucidate the expression,therapeutic effects,and potential mechanisms of ACSL1 in ccRCC through clinical cases,in vitro experiments,nude mouse xenograft models,and bioinformatics analysis.Through comprehensive research,the goal is to provide theoretical basis for ACSL1 as a potential therapeutic target for ccRCC.Objective1.Clarify the relationship between ACSL1 and clinical parameters in ccRCC;2.Investigate the effect of ACSL1 on the proliferation and migration ability of ccRCC cells;3.Study the potential mechanisms by which ACSL1 inhibits the proliferation of ccRCC.MethodsPart 1 Expression of ACSL1 in Clear Cell Renal Cell Carcinoma and Its Correlation with Clinical Features1.Analysis of ACSL1 Expression Levels in ccRCC:Conducting ACSL1 expression analysis using the TCGA-KIRC dataset and two ccRCC datasets,GSE6344 and GSE36895,obtained from the public GEO database;Comparative analysis of clinical samples:Collecting postoperative ccRCC and adjacent normal tissue samples from clinical patients and validating the differential expression and cellular localization of ACSL1 in ccRCC tumor tissues and adjacent normal tissues through western blotting,RT-q PCR,frozen section immunofluorescence,and paraffin section immunohistochemical staining;2.Analysis of Clinical Characteristics:Analyzing the correlation between ACSL1 m RNA expression levels and ccRCC molecular subtypes,pathological grades,lymph node metastasis status,and clinical prognosis;3.ROC Curve Analysis:Evaluating the potential application of ACSL1 in ccRCC diagnosis,including assessing its effectiveness as a diagnostic criterion.Part 2 Overexpression of ACSL1 Inhibits Proliferation and Migration Ability of Clear Cell Renal Cell Carcinoma Cells1.Construction of ccRCC Cell Lines Overexpressing ACSL1:Using lentiviral vectors to construct ACSL1 overexpressing A498 and 786O renal cancer cell lines.ACSL1 m RNA and protein levels were detected using RT-PCR and western blotting techniques;2.In vitro experiments:Assessing the impact of ACSL1 on the viability and proliferation of ccRCC cells through 7-AAD flow cytometry,CCK8 proliferation assays,and colony formation assays;Evaluating the effect of ACSL1 on the migration ability of ccRCC cells through scratch wound healing assays;3.In vivo experiments:Exploring the effect of ACSL1 on the in vivo growth of ccRCC cells through subcutaneous tumor formation in mouse models.Part 3 Overexpression of ACSL1 Induces Iron Death in Clear Cell Renal Cell Carcinoma Through the p53/SLC7A11 Signaling Pathway1.Treatment of A498 and 786O cells with ferroptosis inhibitors（Liproxstatin-1 and Ferrostatin-1）,apoptosis inhibitors（Z-VAD-FMK）,necrosis inhibitors（Necrostatin-1）,and autophagy inhibitors（3-MA）to determine the main type of cell death after ACSL1overexpression;2.Observation of mitochondrial structural changes in ccRCC cells after ACSL1overexpression using transmission electron microscopy;3.Evaluation of ferroptosis-related indicators:Detection of key proteins SLC7A11 and GPX4 levels using western blotting;Systematic evaluation of changes in ferrous ions,ROS,GSH,mitochondrial membrane potential,and lipid peroxidation markers MDA and C11-BODIPY in in vitro and in vivo experimental models;4.Non-targeted lipidomics and transcriptomics to detect changes in lipid and transcription levels after ACSL1 overexpression in A498 cell lines,determining downstream signaling pathways;5.Detection of changes in p53 protein levels in ccRCC cells after ACSL1 overexpression using western blotting;Treatment of cells with p53-specific inhibitor pifithrin-βand detection of changes in p53,SLC7A11,and GPX4 proteins using western blotting;Systematic evaluation of changes in cell viability,ferrous ions,ROS,GSH,mitochondrial membrane potential,and lipid peroxidation markers MDA and C11-BODIPY in in vitro experimental models to determine whether ACSL1 activates ferroptosis by upregulating p53 and inhibiting the SLC7A11/GPX4 signaling pathway;6.Treatment of cells with ROS scavenger NAC to detect changes in p53 protein levels using western blotting,while also examining the aforementioned ferroptosis-related indicators to determine whether ROS affects p53 expression and downstream ferroptosis levels in ACSL1-overexpressing cells.ResultsPart 1 Expression of ACSL1 in Clear Cell Renal Cell Carcinoma and Its Correlation with Clinical Features1.Compared with adjacent normal renal tissue,the m RNA and protein levels of ACSL1were significantly decreased in ccRCC tissues;2.ACSL1 was closely associated with various clinical features of ccRCC:Downregulation of ACSL1 m RNA expression was associated with poorer prognosis,higher cc B molecular subtype,higher pathological grade,and lymph node metastasis;3.Prognostic analysis:Bioinformatics analysis showed that high expression of ACSL1was closely related to better prognosis in patients;4.ACSL1 demonstrated significant diagnostic capability,effectively distinguishing between normal and tumor tissues.Part 2 Overexpression of ACSL1 Inhibits Proliferation and Migration Ability of Clear Cell Renal Cell Carcinoma Cells1.Both ACSL1 m RNA and protein levels were significantly increased in ccRCC cell lines overexpressing ACSL1,confirming effective transfection;2.In vitro experiments:ACSL1 overexpression significantly increased the proportion of7-AAD-positive cells,reduced cell proliferation rate,and inhibited colony formation and migration ability of ccRCC cell lines,indicating that ACSL1 could suppress the viability,proliferation,and migration ability of ccRCC cells;3.In vivo experiments:The growth rate and final tumor weight of tumors in ACSL1-overexpressing group mice were significantly lower than those in the control group,confirming the anti-cancer effect of ACSL1 in vivo.Part 3 Overexpression of ACSL1 Induces Iron Death in Clear Cell Renal Cell Carcinoma Through the p53/SLC7A11 Signaling Pathway1.Determination of cell death type:Treatment with iron death inhibitors significantly inhibited ACSL1-induced cell death in ccRCC cells;ACSL1 overexpression led to decreased expression of SLC7A11 and GPX4 proteins;Mitochondrial volume reduction and decrease or disappearance of mitochondrial ridges were observed;Levels of ferrous ions,MDA,ROS,and C11-BODIPY were upregulated,while GSH and mitochondrial membrane potential were decreased;Treatment with iron death inhibitors effectively restored the above indicators;These results indicate that ACSL1 overexpression induces significant iron death in ccRCC cells;2.In vivo experiments also confirmed upregulation of ferrous ions,MDA,ROS,and C11-BODIPY levels and downregulation of GSH levels in tumors overexpressing ACSL1;Decreased expression of SLC7A11 and GPX4 proteins further supported the evidence of iron death occurrence;3.Multi-omics analysis:Lipidomics analysis suggested ACSL1 overexpression led to changes in lipid metabolism related to iron death,particularly activation of the arachidonic acid metabolism pathway;Transcriptomics analysis found ACSL1 affected various cell death modes and activation of the p53 signaling pathway,revealing the possible mechanism by which ACSL1 promotes iron death through the p53 pathway;4.ACSL1 overexpression increased p53 levels,inhibited SLC7A11 and GPX4expression,and significant alleviation of ACSL1-induced iron death was observed with p53inhibitor;5.ACSL1 promoted p53 activation by increasing ROS,forming a positive feedback loop for iron death,and significant reduction of ACSL1-induced iron death indicators was observed with ROS scavenger.Conclusion1.The expression level of ACSL1 in ccRCC is significantly reduced,negatively correlated with pathological grading and lymph node metastasis status,while positively associated with a better prognosis;2.Overexpression of ACSL1 significantly promotes cell death in ccRCC cells and effectively inhibits cell proliferation capability;3.ACSL1 triggers cellular ferroptosis by enhancing the expression of p53 protein,thereby inhibiting the SLC7A11/GPX4 pathway;4.Overexpression of ACSL1 induces ferroptosis,leading to the accumulation of ROS,which in turn upregulates the expression of p53 protein,forming a positive feedback loop and further activating ferroptosis.