Sphingolipid Metabolism Related Gene IRX3 Promotes Apoptosis Of Melanoma Cells And Influences Prognosis Through PI3K/AKT Signaling Pathway

Background: Melanoma,known as skin cutaneous melanoma(SKCM),is a deeply invasive tumor originating from melanocytes.Its high incidence,recurrence rate,low diagnostic rate,and low cure rate pose significant challenges to clinical physicians.The etiology of melanoma remains obscure,and there is still a lack of prompt,reliable diagnostic methods and safe,effective treatment approaches in clinical practice.Sphingolipids play a critical role in maintaining cellular integrity and functionality and engage in diverse biological processes such as intercellular communication,cell differentiation,and proliferation.Furthermore,bioactive sphingolipids have been identified as vital participants in the highly regulated process of cell death,with extensive research conducted on their cellular activities.Essential enzymes involved in sphingolipid biosynthesis encompass ceramide synthases(Cers),sphingomyelinases(SMase),sphingosine kinase 1(SK1),and sphingosine kinase 2(SK2),with their varying activities occurring across different cellular subcompartments like mitochondria,plasma membrane,or lysosomes.These enzymatic activities can affect different mechanisms of cell death,including apoptosis and necrosis.Tumor immunotherapy has also introduced novel strategies for melanoma treatment,including immune checkpoint therapy,adoptive immunotherapy,tumor vaccine therapy,albeit their application is still in the preliminary stages for melanoma.Recent studies have unveiled lipid metabolism abnormalities to underpin the regulation of various malignant phenotypes in tumors,although the precise role and mechanisms of sphingolipid metabolism in melanoma remain elusive.Aims: Our aim is to investigate and identify biomarkers based on sebaceous-related genes(SRGs)that can be used to predict the response of melanoma patients to immunotherapy and understand its underlying mechanisms.We also aim to develop a prognostic model to predict the prognosis of melanoma patients and explore the specific cellular and biological mechanisms involved.Methods: We obtained single-cell sequencing data of melanoma tissue from the GEO database(No.GSE123139).The data underwent cleaning,filtering,and selection before establishing a prognostic model associated with SRGs.We constructed a clinical nomogram and performed univariate and multivariate analyses to identify the hub gene IRX3.We also evaluated the immune landscape of melanoma and predicted patient responsiveness to immunotherapy.Subsequently,we collected a total of 20 pairs of melanoma and adjacent tissue specimens from patients.Protein and RNA samples were extracted from both melanoma and adjacent tissues,and embedded sections were prepared.WB,q RT-PCR,and IHC were performed to identify differences in protein and m RNA expression levels of the IRX3 gene between tumor and normal tissues.We cultured two melanoma cell lines,WM-115 and A375,in vitro.The IRX3 gene was knocked down using transfection technology,and its effects on tumor cell proliferation,invasion,and migration were evaluated through scratch,CCK8,colony formation,and Transwell assays.Apoptosis-related proteins(Bax,Bcl-2,Cleaved caspase-3)were detected by flow cytometry,apoptosis assay kits,and WB to verify the impact of IRX3 knockdown on tumor cell apoptosis.The expression of key proteins in the PI3K-AKT signaling pathway(p AKT(Thr308),p AKT(Ser473),p-PI3K(Tyr458),total AKT,total PI3K)was measured by Western blot in normal melanoma cells and the knockdown group.An in vivo tumor growth experiment was conducted to assess the effect of IRX3 changes on the development and lung metastasis of melanoma.Additionally,a rescue experiment was performed to investigate whether the tumor suppressive effect induced by IRX3 knockdown was weakened when a PI3 K activator(740Y-P)was added.Results: After quality control,a total of 2,725 high-quality cells were collected from single-cell RNA sequencing data for further study.UMAP method was used to perform dimensionality reduction clustering and visualization of the sample cells based on the gene expression differences,resulting in the identification of 14 cell subgroups.Four genes(PSAP,APOE,ASAH1,and DEGS1)related to sphingolipid metabolism were selected as marker genes to perform further re-clustering analysis of high sphingolipid metabolism cells,which resulted in the subdivision of the high sphingolipid metabolism cell group into 7 classes.The enrichment pathways of the marker genes and various differentially expressed genes in the 7 cell classes were analyzed.Next,the ss GSEA scores of the top 10 marker genes in each cell class were calculated,and based on these scores,melanoma patients were divided into high and low-risk groups.Prognostic analysis showed that the higher the proportion of the 7 highly expressed sphingolipid metabolism cells,the worse the prognosis.Univariate Cox regression analysis was then used to identify genes associated with the survival of melanoma patients,and these genes were further subjected to LASSO Cox regression analysis to select 11 genes(IRX3,PLA2G2 D,GBP1P1,FCGR2 A,GALM,FERMT3,IGKJ5,IL15,IDO1,CMAHP,and HIVEP3)for constructing a risk model.Subsequent validation of this model showed good specificity and accuracy in predicting the clinical prognosis of melanoma patients,and it can provide predictive data for immunotherapy response in melanoma patients.Immune correlation analysis showed that protective genes(GBP1P1,FCGR2 A,GALM,FERMT3,IGKJ5,IL15,IDO1,CMAHP,HIVEP3)were positively correlated with immune response,while the risk gene IRX3 was negatively correlated.CD8 positive T cells,activated state CD4 positive memory T cells,activated state NK cells,and M1 macrophages were positively correlated with model genes,while resting state CD4 positive memory cells,resting state NK cells,M0,and M2 macrophages were negatively correlated with various model genes.Pathway enrichment analysis results showed that except for IRX3,the model genes were positively correlated with immune-related pathways such as JAK-STAT signaling pathway,intestinal immune network,B cell receptor signaling pathway,toll-like receptor signaling pathway,etc.Western blot on 20 pairs of melanoma and adjacent normal tissues showed that the protein expression of IRX3 was upregulated in melanoma tissues,and q RT-PCR tests on 12 pairs of melanoma and adjacent normal tissues showed that the m RNA expression of IRX3 was upregulated in melanoma tissues.IRX3 was knocked down in A375 and WM-115 cell lines,and q PCR and Western blot confirmed the silencing efficiency,with significantly downregulated protein and m RNA levels of IRX3 in the knockdown groups,confirming the successful construction of knockdown cell lines.In colony formation assay,we observed that the ability of melanoma cells with suppressed IRX3 expression to form cell colonies was significantly inhibited.In contrast,melanoma cells with normal expression of IRX3 formed more and larger colonies.In CCK-8 assay,the proliferation rate of melanoma cells was significantly decreased when IRX3 expression was inhibited,and compared with cells with normal expression of IRX3,the absorbance values of cells with inhibited IRX3 expression were lower at each time point,indicating a significant inhibition of their proliferation capacity.Scratch assay results showed that the scratch healing degree of cells with knockdown of IRX3 was reduced at 48 hours compared with the control group,indicating a decrease in the migration ability of cells with knockdown of IRX3.Transwell assay results showed that the silencing of IRX3 significantly inhibited the ability of A375 and WM-115 cells to penetrate the chamber.This indicates that after silencing IRX3 in A375 and WM-115 cells,the proliferation,invasion,and migration abilities of melanoma cells were significantly reduced.Flow cytometry and apoptosis assay kit detection showed that after knocking out IRX3,the proportion of apoptotic cells in A375 and WM-115 cells increased significantly.Western blot showed that knocking out IRX3 reduced the level of anti-apoptotic protein Bcl-2,increased the levels of pro-apoptotic proteins Bax and Cleaved caspase-3,and increased cell apoptosis.Western blot detected that the protein expression levels of p AKT(Thr308),p AKT(Ser473),and p-PI3K(Tyr458)were reduced in A375 and WM-115 cells after silencing IRX3,while there was no statistically significant difference in the expression levels of total AKT and total PI3 K,indicating that the PI3K-AKT signaling pathway was inhibited.In the subcutaneous tumor experiment and immunohistochemical staining results,it was shown that the tumor inhibitory effect induced by silencing IRX3 was partially weakened when PI3 K activator 740Y-P was added simultaneously.In the stable transfected luciferase A375 cell line tail vein injection lung metastasis model in vivo imaging experiment,it was found that silencing IRX3 inhibited the lung metastasis ability of A375 cells.Addition of 740Y-P partially reversed this inhibitory effect.Conclusion:1.The risk model based on SRGs accurately predicts the prognosis of melanoma patients and provides predictions of the effectiveness of immunotherapy.2.Clinical melanoma patients exhibit an increased expression of IRX3 compared to adjacent normal tissue.3.IRX3 plays a significant role in the malignant behavior of melanoma cells,and knocking down IRX3 can improve the malignant behavior of melanoma.4.IRX3 plays an essential role on melanoma cells by suppressing apoptosis.5.IRX3 regulates growth and proliferation in melanoma through the PI3K-AKT signaling pathway.6.Silencing IRX3 inhibits the lung metastasis ability of melanoma A375 cell line.7.The IRX3 gene may serve as a potential therapeutic target for melanoma patients.