Establishment of potential lncRNA-related hub genes involved competitive endogenous RNA in lung adenocarcinoma

Abstract

Long non-coding RNAs (lncRNAs) have a notable role in the diagnosis and prognosis of cancer. However, the associations between lncRNA-related hub genes (LRHGs) expression and the corresponding outcomes have not been fully understood in lung adenocarcinoma (LUAD). Here, a total of 71 patients diagnosed with LUAD and 60 healthy volunteers at The First Affiliated Hospital of Huzhou University from April, 2023 to December, 2023 were enrolled in the present study. A LRHGs model was established using least absolute shrinkage and selection operator analyses of The Cancer Genome Atlas-LUAD datasets. The underlying mechanisms of the LRHGs were investigated via Gene Set Enrichment Analysis and Gene Set Variation Analysis. Additionally, the diagnostic role of serum HOXD cluster antisense RNA 2 (HOXD-AS2) was assessed by receiver operating characteristic (ROC) curve analysis. Lastly, TCGA-LUAD samples were divided into high- and low-HOXD-AS2 expression groups based on the median expression. The associations between HOXD-AS2 expression and miR-4538 as well as Calmodulin-Dependent Protein Kinase Type II subunit Beta (CAMK2B) levels were conducted through Pearson correlation analysis. A comprehensive analysis identified 141 differentially expressed lncRNAs between 539 LUAD tissues and 59 normal samples. A prognostic marker for overall survival was established by constructing a predictive signature consisting of 9 LRHGs. Subsequently, 474 LUAD samples were categorized into a high or low-risk group based on the median of the risk score. An independent prognostic model was constructed to confirm the validity of this categorization. Further comparisons of the clinicopathological features and LRHG-related pathways were performed between the two groups. Examinations of LRHG expression in two LUAD clusters and of the association between LRHG expression and immune infiltration were also conducted. HOXD-AS2 expression was shown to be elevated in LUAD tissues compared with matched normal tissues, and the serum HOXD-AS2 level was also notably increased in LUAD samples compared with healthy controls. The results of the ROC analysis indicated that the sensitivity and specificity of HOXD-AS2 were higher than that of cytokeratin-19 fragment (CYFRA21-1), which is a serum marker for LUAD. Pearson analyses indicated that miR-4538 level was negatively associated with HOXD-AS2 expression, but CAMK2B level showed positive correlation in LUAD. The results of the present study therefore indicated that the constructed LRHG model, particularly HOXD-AS2, could independently diagnose and predict the prognosis of LUAD, which suggested the underlying mechanism of the HOXD-AS2/miR-4538/CAMK2B, and might offer efficient strategies for LUAD treatment.

Keywords: Diagnosis; LncRNA; Lung adenocarcinoma; ceRNA.

Fig. 1

Schematic diagram of the present study. TCGA, The Cancer Genome Atlas; LUAD, lung adenocarcinoma; LASSO, least absolute shrinkage and selection operator; lncRNA, long non-coding RNA; LRHGs, lncRNA-related hub genes; ROC, receiver operating characteristic; KM, Kaplan–Meier; DEGs, differentially expressed genes; GSEA, Gene Set Enrichment Analysis; GSVA, Gene Set Variation Analysis

Fig. 2

Construction of a LRHGs model. A Volcano plot of the LRHGs in TCGA-LUAD datasets. B LRHGs model diagram following LASSO regression. C Variable trajectory diagram of the LASSO regression. D Risk factor map for the LRHGs model. TCGA, The Cancer Genome Atlas; LASSO, least absolute shrinkage and selection operator; LUAD, lung adenocarcinoma; LRHGs, long non-coding RNA-related hub genes

Fig. 3

Prognosis of the LRHGs. A Expression of the LRHGs in TCGA-LUAD datasets. B Diagnostic value of AC100781.1 and AC110285.1. C Diagnostic value of AL596087.2 and HOXD-AS2. D Receiver operating characteristics curves for evaluating the diagnostic value of the LRHGs model in the high and low-risk groups at 1, 3, and 5 years. E The survival curves of patients with LUAD in the high and low-risk groups. The association between (F) AC004687.1, G AC074011.1, H AC110285.1, I AL596087.2, J AP004609.1, or K HOXD-AS2 expression and OS of patients with LUAD. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant. LUAD, lung adenocarcinoma; TCGA, The Cancer Genome Atlas; KM, Kaplan–Meier; OS, overall survival; LRHGs, long non-coding RNA-related hub genes; ROC, receiver operating characteristic; AUC, area under the curve; HOXD-AS2, HOXD cluster antisense RNA 2

Fig. 4

Clinical significance of the LRHGs. Percentage weight of (A) T stage, (B) N stage, (C) M stage, (D) stage, (E) age, (F) sex, (G) OS, (H) DSS and (I) PFI between the high and low-risk groups. TCGA, The Cancer Genome Atlas; OS, overall survival; DSS, disease-specific survival; PFI, progression-free interval

Fig. 5

Construction of a LRHGs prognostic model. A Univariate Cox regression analysis. B A nomogram of the LRHGs prognostic model. Calibration curves for C 1-year, D 3-year and E 5-year. DCA of (F) 1-year, (G) 3-year and (H) 5-year. TCGA, The Cancer Genome Atlas; LUAD, lung adenocarcinoma; LRHGs, long non-coding RNA-related hub genes; DCA, decision curve analysis

Fig. 6

GSEA and GSVA. A GSEA. B Intergin 4 pathway. C Advanced glycosylation end product receptor signaling. D Mitochondrial translation. E Fatty acid metabolism. F Heatmap of the top 20 pathways following GSVA. G Comparison curve of the GSVA. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant. GSEA, Gene Set Enrichment Analysis; GSVA, Gene Set Variation Analysis

Fig. 7

Construction of a lncRNA-RBP interaction network. LncRNA, long non-coding RNA; RBP, RNA-binding protein

Fig. 8

Identification of LUAD-associated subtypes. A Consistent clustering graph (k = 2). B CDF curve of consistent clustering. C Delta curve of the CDF. D Heatmap of the LRHGs expression in clusters 1 and 2. E PCA of the LRHGs in clusters 1 and 2. F Comparison of the LRHGs expression in clusters 1 and 2. G Stromal score. H Immune score. I Estimate score. J Tumor purity. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant. TCGA, The Cancer Genome Atlas; LUAD, lung adenocarcinoma; LRHGs, long non-coding RNA-related hub genes; CDF, Cumulative Distribution Function; PCA: Principal Component Analysis

Fig. 9

Immune infiltration analysis. A Immune infiltration analysis using single sample Gene Set Enrichment Analysis. B Correlation analysis between the infiltration abundance of 28 immune cells and other immune cells. C Correlation analysis between the infiltration abundance of 28 immune cells and expression of the LRHGs. D Immune infiltration analysis using CIBERSORT. E Correlation analysis between the infiltration abundance of 15 immune cells and other immune cells. F Correlation analysis between the infiltration abundance of 15 immune cells and expression of the LRHGs. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant. LRHGs, long non-coding RNA-related hub genes; CIBERSORT, cell type Identification By Estimating Relative Subtypes Of RNA Transcripts

Fig. 10

Verification of the LRHGs expression level. A RT-qPCR analysis of the mRNA levels of the LRHGs in 21 paired LUAD tumor and normal tissues. B RT-qPCR analysis of the HOXD-AS2 mRNA level in 21 paired LUAD tumor and normal tissues. C KM analysis of the overall survival data from TCGA-LUAD datasets containing 601 patients. D KM analysis of the recurrence-free survival data from TCGA-LUAD datasets containing 366 patients. ***P < 0.001. KM, Kaplan–Meier; LUAD, lung adenocarcinoma; HOXD-AS2, HOXD cluster antisense RNA 2; TCGA, The Cancer Genome Atlas; LRHGs, long non-coding RNA-related hub genes; RT-qPCR, reverse transcription-quantitative PCR

Fig. 11

Diagnostic value of serum HOXD-AS2 in LUAD. A Reverse transcription-quantitative PCR analysis of the serum HOXD-AS2 mRNA level in LUAD and healthy controls samples. Associations between the serum HOXD-AS2 level and the (B) TNM stage, (C) lymph node metastasis and (D) tumor differentiation of patients with LUAD. E The diagnostic value of serum HOXD-AS2 and CYFRA21-1 in LUAD was measured by receiver operating curve analysis. **P < 0.01, ***P < 0.001. LUAD, lung adenocarcinoma; HOXD-AS2, HOXD cluster antisense RNA 2. CYFRA21-1, cytokeratin-19 fragment

Fig. 12

CeRNA analysis of HOXD-AS2 in LUAD. A Pearson correlation analysis was used to investigate the association between miR-4538 and HOXD-AS2. B A Wayne diagram conducted in conjunction with the TargetScan database, the miRWalk database, the HEmRNAs and the necroptosis-related gene list. C Pearson correlation analysis was used to investigate the association between CAMK2B and HOXD-AS2