Screening of ferroptosis-related lncRNAs in LAUD.
Comparing LAUD tissues with adjacent non-LAUD tissues, 1224 differentially expressed lncRNAs were found, of which 1044 are up-regulated and 180 are down-regulated (Additional file 5). The correlation results between 259 ferroptosis-related genes and differentially expressed lncRNAs shown that there are 195 ferroptosis-related lncRNAs (FRlncRNAs) (Additional file 6).
Construction, validation, and evaluation of an nine ferroptosis-related lncRNAs prognostic signature
The entire set (N=477) with 195 FRlncRNAs expression data was randomized into the test set (N=237) and train set (N=240). In the univariate Cox regression assessment, 22 FRlncRNAs modulated the overall survival of the patients in the train set (Fig. 1a). Lasso regression was used for further analysis to eliminate overfitting lncRNAs, and the 14 lncRNAs we obtained were used for the subsequent multivariate Cox regression analysis (Fig. 1b-d) (concordance index [C-index], 0.75). The ferroptosis-associated lncRNA prognostic biosignature was developed based by summing up the product of each lncRNA expression with its corresponding coefficient in multivariate Cox regression as indicated below: lncRNA biosignature risk score= (0.049× expression of AC099850.3) + (0.060× expression of NAALADL2-AS2) + (0.051× expression of AL844908.1) + (0.056× expression of AL365181.2) + (-0.078× expression of SMIM25) + (0.090× expression of FAM83A-AS1) + (0.090× expression of LINC01116)+ (0.089× expression of AL049836.1)+ (-0.232× expression of C20orf197). Analysis using the Proportional Hazards Assumption in the Cox model revealed that all the P values >0.05, implying they conformed to the PH test (Additional file 7).
According to the median value of the risk score, results of the Kaplan-Meier curves demonstrate that the high-risk group has a remarkably dismal overall survival (OS) in contrast with the low-risk group in the train set (P=8.66E-06), test set (P=2.766E-04), and entire set (P=7.533E-09) (Fig. 2a-c). The train set shows three years' OS for patients with high and low-risk group were 38.3% and 73.3%, respectively. The test set is 41.3% and 79.3%, respectively. The entire set is 40.9% and 78.4%, respectively. The AUC of three years dependent ROC for the seven-lncRNA biosignature achieves 0.754, 0.716, and 0.738 respectively in the train set, test set, and entire set (Fig. 2d-f), which demonstrate the good performance of the model in estimating the LAUD patients' OS. The mortality rate was higher in patients with high-risk scores relative to those with low-risk scores in the three sets (Fig. 2g-i). The seven lnRNAs’ (AC099850.3, NAALADL2-AS2, AL844908.1, AL365181.2, FAM83A-AS1, LINC01116, AL049836.1) expression of signature were lower in low-risk group compared to the high-risk group in cluster heat map, SMIM25 and C20orf197 oppositely (Fig. 2j-l).
It is worth noting that AC099850.3, FAM83A-AS1 and LINC01116's high expression of this lncRNA signature also has a worse OS than low, C20orf197 oppositely (Fig. 3). The association of the seven lncRNAs with ferroptosis genes is shown by network diagram in Fig. 4. In addition, we stratified according to various clinical factors (gender, age, clinical stage, postoperative tumor status, KRAS status, EGFR status, ALK status, ECOG score) and applied the prognostic model to OS detection, which is shown in Fig. 5, the results shown that the signature has good predictive significance for LAUD patients in most stratification factors, and part of results are not satisfactory (P>0.05), which might be due to there are not enough samples in these stratifications.
Independent prognostic analysis of the nine ferroptosis-associated lncRNAs signature and its correlation with clinical variables.
The Univariate Cox regression assessment demonstrated that the lncRNA biosignature risk score was evidently correlated with the patients’ OS (hazard ratio HR=1.003, confidence interval 95%CI=1.001–1.006, P=0.009) (Table 2). Moreover, the multivariate Cox regression analysis demonstrated that the lncRNA biosignature risk score remained independent with OS considering other conventional clinical factors including Lymph-node status, the clinical stage, distant metastasis, and T stage (HR=1.004, 95% CI=1.002–1.007, P=0.001). Meanwhile, clinical stage was demonstrated as an independent prognostic index. Compared to clinical variables, this signature risk score's ROC curves of three years demonstrate the largest AUC value (0.737) (Fig. 6).
Based on the stratification of clinical variables, the correlation between the lncRNAs and clinical variables shows that clinical stage is related to AC099850.3, AL365181.2, FAM83A-AS1, LINC01116, C20orf197’ expression and signature’ risk score. T stage is associated with AC099850.3, FAM83A-AS1, AL049836.1 and C20orf197’ expression and signature’ risk score. Lymph-node status is correlated to AC099850.3, FAM83A-AS1’ expression and signature’ risk score. Distant metastasis is concerning to AL365181.2 (Fig. 7).
Functional enrichment analysis of the nine ferroptosis-related lncRNAs signature.
GSEA analysis is used to discover potential biological functions of the nine ferroptosis-associated lncRNAs signature of LAUD (Fig. 8 and Table 3). The results showed that eight tumor-related and metabolism-related signaling pathways (KEGG_CELL_CYCLE, KEGG_MISMATCH_REPAIR, KEGG_P53_SIGNALING_PATHWAY, KEGG_SMALL_CELL_LUNG_CANCER, KEGG_UBIQUITIN_MEDIATED_PROTEOLYSIS) are obviously enriched in the high-risk group, and three signaling cascades (KEGG_ALPHA_LINOLENIC_ACID_METABOLISM
KEGG_ARACHIDONIC_ACID_METABOLISM, KEGG_FATTY_ACID_METABOLISM) were abundant in the low-risk group by c2.cp.kegg.v7.2.symbols.gmt. These results suggest that this signature model function as LAUD’ prognostic factor through signaling pathways.