| Lung cancer is one of the most common malignant tumors,and it is also the malignant tumor with the highest mortality rate in the world.About 25%of cancer patients die from lung cancer.According to pathological classification,non-small cell lung cancer(NSCLC)is the most common type,accounting for about 85%of all lung cancers,of which lung adenocarcinoma(LUAD)is one of the most common subtypes.At present,surgical resection is still the first choice for the treatment of LUAD.In recent years,molecular targeted therapy and immunotherapy have developed rapidly,bringing hope for the clinical treatment of a series of malignant tumors.However,because lung cancer is a highly heterogeneous tumor with complex molecular mechanisms,the 5-year overall survival(OS)of LUAD patients is still far from satisfactory.Due to the lack of specific technologies that can diagnose cancer at an early stage,there is an urgent need to further discover specific prediction methods for LUAD patients in order to find new therapeutic targets and improve the survival rate of patients.Research on tumor ferroptosis has increased rapidly over the past few decades.This is a recently identified mode of programmed cell death,distinct from other forms of cell death such as apoptosis,necrosis,and autophagy.It is iron-dependent and is characterized by the accumulation of intracellular reactive oxygen species.With the intensive study of ferroptosis,cancer researchers have determined that the induction of ferroptosis plays a key role in killing tumor cells and inhibiting tumor growth.There is increasing evidence that ferroptosis is involved in the biological processes of LUAD.However,the specific mechanism of ferroptosis in the occurrence and development of lung cancer has not been elucidated,and its significance in the treatment and prognosis of lung cancer needs to be further revealed.In addition,studies have shown that the effect of ferroptosis on tumors involves a variety of immune cells and immune factors,and plays a key role in tumor immunity.Therefore,exploring biomarkers related to tumor immunity and ferroptosis is of great significance for the immunotherapy of lung cancer.Long non-coding RNA(lncRNA)are RNAs with a molecular weight of more than 200 nucleotides,which are usually not involved in the translation of proteins,but can play a role in epigenetic regulation,cellular It plays an important role in many processes such as cycle regulation and cell differentiation regulation.Previous studies have shown that the abnormal expression of lncRNAs in LUAD is widely involved in the process of tumor proliferation and metastasis,and ferroptosis-related lncRNAs may be prognostic risk factors,and the constructed model can be used to distinguish high-risk patients from low-risk patients.Currently,we still lack a comprehensive understanding of the role of ferroptosis-related lncRNAs in the prognosis of LUAD,and further exploration of their full roles is required.Therefore,based on the LUAD dataset of The Cancer Genome Atlas(TCGA),we retrieved ferroptosis-related genes from the FerrDb database,and identified potentially deregulated ferroptosis-related lncRNAs and ferroptosis-related genes through bioinformatics analysis.A prognostic model of ferroptosis-related lncRNA was constructed by LASSO regression,and the validity of the model was confirmed by receiver operating characteristic curve(ROC)and Kaplan-Meier survival analysis,and the relationship between the model and tumor immune response was also discussed.correlation.Subsequently,we constructed a nomogram to predict overall survival in patients with LUAD,and used KaplanMeier survival analysis,Decision Curve Analysis(DCA)and ROC curves to test the performance of the nomogram.In order to determine the expression of PACERR and LINC00941,we performed Quantitative Real-time PCR(qRT-PCR).The functions of PACERR and LINC00941 were evaluated by experiments of cell invasion,colony formation and cell proliferation of A549 and H1299 cells.This study explored the significance and molecular mechanism of ferroptosis-related lncRNAs in LUAD through bioinformatics methods,which will help improve the early diagnosis rate of LUAD and provide a theoretical basis for precise and individualized treatment.Part Ⅰ Construction and validation of a prognostic model of ferroptosis-related lncRNA in patients with lung adenocarcinomaObjective:Based on bioinformatics,a ferroptosis-related lncRNA prognostic model was constructed using transcriptome data from the TCGA database to predict the overall survival of patients with LUAD,and to explore the immune cell infiltration,tumor mutational burden(TMB)and signaling pathways related to the model..Based on the prognostic model and clinicopathological data,a nomogram was constructed to predict the individual survival of LUAD patients,and to verify the accuracy and clinical reliability of its prediction of prognosis.Methods:1.Clinicopathological data(stage,sex,age,and follow-up data)and gene expression profiles of LUAD patients were downloaded from the TCGA database.Download the list of ferroptosis-related genes from the FerrDb database.The dataset was randomly divided into training and validation sets using the "caret" package.2.Pearson correlation was used to correlate the expression levels of lncRNAs and ferroptosis-related genes to identify ferroptosis-related lncRNAs(correlation coeffici-ent>0.40,P<0.001).Differential expression analysis of ferroptosis-related genes and ferroptosis-related lncRNA was performed using the "limma" package,with P<0.05 and |log2(foldchange)|>1 as the definition criteria for differential expression.3.Differentially expressed ferroptosis-related genes were subjected to KEGG signaling pathway and GO functional enrichment analysis using the "clusterProfiler"package.4.Univariate Cox regression and LASSO regression were performed on the training set to screen for differentially expressed Ferroptosis-related Differentially Expressed lncRNA(FRDEL)closely related to the survival of LUAD patients,and then used to construct a lncRNA prediction model.The risk score(RS)of each sample was calculated according to the expression of lncRNA and the corresponding regression coefficient,and then,the patients in the training set,validation set and the whole cohort were divided into high-risk group and low-risk group according to the median risk score groups,and Kaplan-Meier survival analysis was performed separately.5.ROC analysis was performed on the training set,validation set and the entire cohort using the "timeROC" package to examine the predictive performance of the FRDEL model.6.The CIBERSORT algorithm was used to measure the proportion of 22 different types of immune cells in each patient,and the differences in the proportion of invading immune cells in the low-risk group and the high-risk group were compared.TMB data were downloaded from the TCGA database,and the TMB for each patient in the high-risk and low-risk groups was calculated using the maftools"package.7.The co-expression network of ferroptosis-related lncRNAs and mRNAs was constructed by Cytoscape software.The ferroptosis-related lncRNAs in low risk group and high risk group were analyzed by GSEA,and the statistical significance was set as FDR<0.25and NOM P-val<0.05 respectively.8.Univariate Cox and multivariate Cox regression analyses were performed on risk scores and clinicopathological data,respectively,and nomograms were constructed using the rms software package to predict one-year,three-year,and five-year survival in patients with LUAD.The predictive performance of the nomogram was examined using ROC curves and decision curve analysis(DCA).Results:1.We obtained expression profiling data for 54 normal tissues and 497 LUAD tissues and 259 ferroptosis-related genes.468 LUAD patients with complete survival data were included in the final analysis and the dataset was randomly divided into training set(n=312)and validation set(n=156).2.2634 ferroptosis-related lncRNAs were identified by Pearson correlation analysis.A total of 909 FRDELs and 71 ferroptosis-related DEGs(23 down-regulated and 48 up-regulated)were identified.3.The GO enrichment analysis of 71 ferroptosis-related DEGs showed that BP was involved in oxidative stress response,reactive oxygen species metabolism,metal ion response,etc.;MF mainly regulated oxidoreductase activity and the binding of molecular oxygen and iron ions.CC is mainly upregulated in the oxidoreductase complex and the apical plasma membrane synthesis pathway.KEGG enrichment analysis showed that the overexpressed ferroptosis genes were mainly involved in ferroptosis and hypoxia-inducible factor-1(HIF-1)signaling pathway.4.A 14-FRDEL model was constructed including 9 risk factors(AP001610.2、AC004832.5、AL355472.3、PACERR、AC007773.1、AC116552.1、AC108451.2、LINC00941 and LINC01638)and 5 protective factors(AC034102.8、AF131215.5、AC026355.2、MIR223HG和AC246787.2),and can accurately distinguish the prognosis of patients with LUAD.The survival curves of the high-risk and low-risk groups were significantly different,with p-values of Logrank test<0.001,0.008,and<0.001,respectively.5.The results of the ROC curve constructed according to the risk score showed that for the OS of LUAD patients,the AUC values of the training set,validation set and the whole cohort were 0.773,0.717 and 0.751,respectively.6.In high risk group,the number of resting mast cells(MCR),CD4 memory T cells and resting natural killer cells(NK cells)increased,while the number of monocytes,M0 and M1 macrophages,active mast cells(MCA)and resting dendritic cells(DC)decreased significantly.KRAS(28%vs.21%),TP53(52%vs.38%),TTN(47%vs.38%),MUC16(44%vs.34%),FLG(25%vs.18%),and ADAMTS12(18%vs.14%)were the most common mutations in high-risk and low-risk groups.In the high risk group,the number of TP53 mutations increased significantly(P=0.047),the frequency of mutations was higher(P=0.002),and the level of TMB increased(P=0.00067).7.The co-expression network of lncRNAs and mRNAs was visualized using Cytoscape software.GSEA analysis showed that the 14-FRDEL model was involved in some cancer-related signaling pathways,such as DNA replication,p53 signaling pathway,and cell cycle.8.A nomogram incorporating the 14-FRDEL model and clinicopathological factors was constructed,and the AUC values for predicting 1-,3-,and 5-year overall survival(OS)probabilities were 0.783,0.761,and 0.774,respectively.DCA shows that the area under the curve represented by the nomogram model is basically larger than the area under the curve represented by other parametric models.Conclusions:1.Based on bioinformatics,a 14-FRDEL model was constructed,including nine risk factors(AP001610.2,AC004832.5,AL355472.3,PACERR,AC007773.1,AC116552.1,AC108451.2,LINC00941 and LINC01638)and five protective factors(AC034102.8,AF131215.5,AC026355.2,MIR223HG and AC246787.2),which may provide accurate predictions for the prognosis of LUAD patients.This model is closely related to some infiltrating immune cells and mutant genes in tumors.2.A nomogram incorporating the 14-FRDEL model and clinicopathological factors was developed to predict survival outcomes in LUAD patients with good predictive performance.This model may be a reliable tool for developing novel ferroptosis-related therapeutic strategies and improve outcomes for patients with LUAD.Part Ⅱ:Experimental study on PACERR and LINC00941 promoting lung adenocarcinoma cell invasion,metastasis and inhibiting apoptosisObjective:The expression levels of PACERR and LINC00941 in LUAD cells were detected,and the effects of PACERR and LINC00941 on the proliferation,apoptosis,invasion and migration of LUAD cells A549 and H1299 were analyzed to further verify the reliability of 14-FRDEL model.Methods:1.The expression levels of PACERR and LINC00941 in LUAD cells were detected by qRT-PCR technique,and the expressions of PACERR and LINC00941 in LUAD cells and normal human lung epithelial cells were compared.2.The effects of PACERR and LINC00941 on the proliferation of LUAD cells A549 and H1299 were analyzed by CCK-8 test and plate cloning test.3.The effects of PACERR and LINC00941 on apoptosis of LUAD cells A549 and H1299 were analyzed by flow cytometry and western blot assay.4.The effects of PACERR and LINC00941 on the invasion and migration of LUAD cells A549 and H1299 were analyzed by scratch test and cell invasion test.Results:1.The results of qRT-PCR showed that the expression of PACERR and LINC00941 in A549,H1792 and H1299 cells was significantly higher than that in MRC-5 cells.2.The results of CCK-8 assay showed that the viability of pcDNA-PACERR and pcDNA-LINC00941 cells in A549 and H1299 cells was significantly higher than that of NC cells at 24 h,48 h,72 h and 96 h,while that of si-PACERR and si-LINC00941 cells was significantly lower than that of NC cells.The results of plate cloning assay showed that in A549 and H1299 cells,the clone formation ability of pcDNAPACERR and pcDNA-LINC00941 cells was significantly higher than that of NC cells,while that of si-PACERR and si-LINC00941 cells was significantly lower than that of NC cells.3.The results of flow cytometry showed that the apoptosis rate of pcDNAPACERR and pcDNA-LINC00941 cells was significantly lower than that of NC cells,while that of si-PACERR and si-LINC00941 cells was significantly higher than that of NC cells.Western blot assay showed that in A549 and H1299 cells,the expression of Bcl-2 in pcDNA-PACERR and pcDNA-LINC00941 cells was significantly higher than that in NC cells,while the expression of Bax and caspase-3 in si-PACERR and si-LINC00941 cells was significantly lower than that in NC cells.The expression of Bcl-2 in si-PACERR and si-LINC00941 cells was significantly lower than that in NC cells,while the expression of Bax and caspase-3 was significantly higher than that in NC cells.4.The results of scratch assay showed that in A549 and H1299 cells,the migration rate of pcDNA-PACERR and pcDNA-LINC00941 cells was significantly higher than that of NC cells,while that of si-PACERR and siL-INC00941 cells was significantly lower than that of NC cells.The results of Transwell chamber experiment showed that the number of penetrating cells of pcDNA-PACERR and pcDNA-LINC00941 in A549 and H1299 cells was significantly higher than that of NC cells,while the number of transmembrane cells of si-PACERR and si-LINC00941 was significantly lower than that of NC cells.Conclusions:1.Compared with normal human lung epithelial cells,PACERR and LINC00941 were up-regulated in LUAD cells,confirming the reliability of the 14-FRDEL model.2.PACERR and LINC00941 can promote the proliferation,invasion and migration of LUAD cells and inhibit the apoptosis of LUAD cells. |
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