Potential Prognostic Roles Of Ferroptosis-related MRNAs And LncRNAs In Bladder Cancer

Part Ⅰ:Potential prognostic roles of Ferroptosis-related mRNAs in bladder cancerObjectiveFerroptosis is a new cell death mode which is different from traditional apoptosis and necrosis.It is reported to play important regulatory roles in the pathogenesis of various malignant cancers.However,the potential relationships between ferroptosis and bladder cancer still keep unclear.This study aims to preliminarily explore whether Ferroptosis-related mRNAs play a potential prognostic role in bladder cancer,and further verify the vital target molecule in vitro.This study aims to identify whether the ferroptosis induction can provide a new direction for the new treatment of bladder cancer after chemotherapy and immunotherapy.MethodsThis study firstly utilized the advantages of the high-throughput second-generation sequencing technology to systematically analyze and verify the mRNA transcriptome matrix of bladder cancer.Depending on differentially expressed ferroptosis-related mRNAs(DEGs)between tumor and normal tissues and the prognostic ferroptosisrelated mRNAs,the optimal ferroptosis-related mRNA prognosis model was constructed by utilizing the least absolute shrinkage and selection operator cox regression(LASSO)analysis.The Kaplan-Meier analysis,univariate cox regression analysis and multivariate cox regression analysis were conducted to further verify the prognostic roles of optimal risk model.The gene ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis,and single sample Gene Set Enrichment Analysis(ssGSEA)were performed to explore potential biological and immunological mechanisms involved in ferroptosis-related mRNAs during bladder cancer samples.The study further screened out targeted ferroptosis-related mRNA molecule and validate it by experiments in vitro.Quantitative real-time polymerase chain reaction(RT-qPCR)and western blot(WB)experiments were performed to verify the expressions of target ferroptosis-related genes between typical bladder cancer cell lines(HT-1376,BIU-87,T24)and normal bladder immortalized epithelial cells(SVHUC-1).Short hairpin RNA(shRNA)was constructed to knock down the expressions of target gene in bladder cancer cells.Its potential cell function was further verified by CCK cell proliferation assay,clone formation assay and the wounding healing assay.At the same time,the mitochondrial morphology of bladder cancer cells was identified by transmission electron microscopy.Furthermore,this study collected clinical bladder cancer samples from Beijing hospital for immunohistochemical staining to verify the detailed expressions of target ferroptosis-related gene in this study.The correlation between target Ferroptosis-related genes and the clinical stages was also further explored in this study.The research further explored the potential relationships between the target mRNA and the immune microenvironment of bladder cancer by using the ESTIMATE analysis and CIBERSORT immune infiltration analysis.ResultsIn this study,totally 33 ferroptosis-related mRNAs were differentially expressed between bladder cancer tissues and normal tissues.(p<0.05)Only eight ferroptosisrelated mRNAs were identified to pose significant prognostic-roles for overall survivals of bladder cancer samples.(p<0.05)The intersection of prognostic ferroptosis-related genes and differentially expressed genes identified 4 prognostic targets,including ALOX5,FANCD2,HMGCR and FADS2.The least absolute shrinkage and selection operator cox regression successfully built a 4-gene signature:risk score value=esum(each gene’s normalized expression*each gene’s coefficient).Kaplan-Meier survival analysis found that a high Ferroptosis-related mRNA risk predicted a significantly poor survival prognosis both in the derivation cohort and the external validation cohort.(p<0.05)Multivariate Cox regression analysis in the derivation cohort identified age(p<0.001),grade(p=0.129)and risk score(p=0.016)as independent prognostic predictors for overall survivals.Multivariate Cox regression analysis in the validation cohort also identified age(p=0.002),stage(p<0.001)and risk score(p=0.006)as independent prognostic predictors for overall survivals.Further analysis showed that the ferroptosis-related mRNA risks were significantly correlated with the molecular subtypes of bladder cancers and also the enrichment levels of various immune cells and immune activities.The vitro experiments further demonstrated that the key ferroptosisrelated gene in the target model,FADS2,showed significantly higher expressions in cancer cell lines than normal bladder cells in this study.(p<0.05)CCK8 cell proliferation assay,clone formation assay and the wounding healing assay showed that the proliferation and migration activity of bladder cancer cells were significantly affected after targeted knockdown of FADS2.Bladder cancer cells were further fixed,dehydrated,embedded,sectioned,and stained.With the help of transmission electron microscopy,it was observed that mitochondria were significantly reduced and the mitochondrial membrane density was significantly increased in knockdown cancer cells.In addition,immunohistochemical staining revealed that FADS2 was significantly overexpressed in bladder cancer tissues compared with normal bladder tissues,and its expression level was significantly correlated with the clinical stages of bladder cancer.(p<0.01).Furthermore,with the help of FADS2 expressions and a variety of clinical elements,this study successfully constructed a clinical prediction nomogram for bladder cancer.Finally,the results of ESTIMATE and CIBERSORT analysis suggested that FADS2 might play a regulatory role in bladder cancer through the tumor immune microenvironment mechanisms.ConclusionFerroptosis-related mRNAs posed excellent predictive-abilities for overall survivals of bladder cancers.Ferroptosis induction could provide new ideas and research directions for the future treatment of bladder cancers.Part Ⅱ:Potential prognostic roles of Ferroptosis-related lncRNAs in bladder cancerObjectivesThe role of ferroptosis-related long non-coding RNAs(lncRNAs)in bladder cancer remains elusive.This study aims to examine the prognostic roles of ferroptosis-related lncRNAs in bladder cancer.MethodsThe transcriptomic matrix and clinical information of patients with bladder cancer were obtained from The Cancer Genome Atlas(TCGA)database.A ferroptosis-related lncRNA signature was developed via least absolute shrinkage and selection operator(LASSO)analysis using data from the training cohort,and the signature was further validated using data from the validation cohort.The role of AC006160.1,the most significant lncRNA in the risk signature,was examined in various cell lines including SV-HUC-1,BIU-87,HT-1376,T24,RT4,RT-112,5637 and UMUC3.The pcDNA3.1AC006160.1 plasmid was constructed and transfected into the bladder cancer cell lines T24 and BIU-87.In addition,cell proliferation,colony formation,transwell and wound healing assays were performed to examine the biological function of AC006160.1 in T24 and BIU-87 cell lines.Furthermore,this study continued to explore the potential therapeutic responses of high-or low-AC006160.1 group to different therapeutic drugs by utilizing drug sensitivity analysis.ResultsTwo clusters were identified through consensus clustering based on prognostic ferroptosis-related lncRNAs.A 5-lncRNA risk signature was successfully constructed using data from the training cohort and validated using data from the validation cohort.The risk signature had excellent ability to predict survival outcomes,clinical stages,pathological grades,expression of immune checkpoints and immunotherapeutic responses in bladder cancer samples.Furthermore,AC006160.1 expression was found to be lower in the cancer cell lines BIU-87,T24,RT4,RT-112 and 5637 than in the normal control cell line SV-HUC-1.Cell proliferation,colony formation,transwell migration and wound healing assays validated that overexpression of AC006160.1 significantly inhibited the proliferation and invasion abilities of both T24 and BIU-87 cells.Drug sensitivity analysis revealed that patients with high expression of AC006160.1 were sensitive to metformin and methotrexate,and the results were further validated via in vitro drug experiments.ConclusionFerroptosis-related lncRNAs play a vital role in predicting the multiomic characteristics of bladder cancer.The lncRNA AC006160.1 serves as a protective factor for the development of bladder cancer.