| Objective:Cervical cancer as the second most common gynecological cancer in developing countries,has the characteristics of limited treatment options,poor prognosis and high recurrence rate.Ferroptosis-related long non-coding RNAs model was constructed by bioinformatics analysis to improve the ability to predict the prognosis of patients with cervical cancer and to verify the effect of ferroptosis on M1 macrophage polarization and lnc AC022382.1 expression.Methods:1.The clinical,pathological data and gene expression profiles of patients with cervical cancer were downloaded based on The Cancer Genome Atlas.The genes related to ferroptosis were downloaded from Ferr Db database.Pearson correlation was used to evaluate the expression level of lnc RNAs and ferroptosis related genes in order to determine ferroptosis related lnc RNAs.The correlation coefficient was more than 0.3and P < 0.001.Using "limma" package to analyze the differential expression of ferroptosis related genes and ferroptosis related lnc RNAs,P < 0.05 and | log2(fold change)| > 1 was considered significant difference in expression.2.The differentially expressed genes related to ferroptosis were analyzed by Kyoto Encyclopedia of Genes and Genomes signal pathway and gene ontology functional enrichment analysis using "cluster Profiler" package.3.ferroptosis-related lnc RNAs was analyzed by univariate COX regression and multivariate COX regression to screen the differentially expressed ferroptosis-related lnc RNAs which was closely related to the prognosis of cervical cancer patients and to construct an lnc RNAs evaluation model.The risk score of each sample was calculated according to the expression of lnc RNAs and the corresponding regression coefficient,and the cohort patients were divided into high risk group and low risk group according to the median risk score,and survival analysis was carried out by Kaplan-Meier.4.ROC analysis and decision curve analysis were performed to evaluate the prognostic characteristics of cervical cancer and its sensitivity and specificity compared with other clinicopathological features by using "time ROC" software package.5.The co-expression network of ferroptosis-related lnc RNAs and m RNAs was constructed by Cytoscape software.At the same time,ferroptosis-related lnc RNAs in high and low risk groups was analyzed by GSEA to evaluate the difference of biological enrichment.The statistical significance was set as false discovery rate< 0.05.6.CIBERSORT,TIMER and other algorithms were used to compare the differences in the proportion of immune cells,immune response and immune checkpoints between the high risk group and the low risk group.7.The effects of different concentrations of ferroptosis inducer erastin on the secretion of TNF-α by M1 macrophages were analyzed by ELISA.8.The expression of lnc AC022382.1 in Hela cells co-cultured with different concentrations of ferroptosis inducer erastin was detected by q RT-PCR Results:1.The expression profile data of 3 normal tissues and 286 cervical cancer tissues and382 ferroptosis related genes were obtained.2.Based on multivariate Cox regression analysis,18 ferroptosis-related lnc RNAs were selected to predict the prognosis of patients with cervical cancer.Kaplan-Meier analysis showed that the prognosis of patients in low risk group was better than that in high risk group.The AUC value of the model is 0.836,indicating that it can be used to predict the prognosis of cervical cancer.3.GSEA enrichment analysis showed that genes related to immune and metabolic pathways were enriched in low risk group,while nucleotide and protein synthesis and metabolism pathways were more enriched in high risk group.ss GSEA found that there were significant differences in cytolysis,human leukocyte antigen,proinflammatory factor,T cell costimulation and co-inhibition between low risk group and high risk group.Immune checkpoints such as CD-48,CD27 and TNFRSF14 were also expressed differently between the two groups.4.The results showed that after the Hela cells treated with different concentrations of ferroptosis inducer were co-cultured with M 1 macrophages of human or mice,the TNF-α secreted by M 1 macrophages increased significantly with the ferroptosis level of Hela cells,and the expression of lnc AC022382.1 in Hela cells increased with the concentration of ferroptosis inducer erastin.Conclusion:1.In this study,the risk assessment model based on 18 ferroptosis-related lnc RNAs can provide a certain predictive value for the prognosis of cervical cancer.2.The secretion level of TNF-α in M 1 macrophages of human and mice was positively correlated with the degree of ferroptosis in Hela cells,suggesting that the polarization regulation of M 1 macrophages may be a clue of the effect of ferroptosis on the immune microenvironment of patients with cervical cancer.3.Lnc AC022382.1 participates in the process of ferroptosis in Hela cells and plays a role in promoting ferroptosis. |
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