Network analysis of differentially expressed smoking-associated mRNAs, lncRNAs and miRNAs reveals key regulators in smoking-associated lung cancer

Abstract

Lung cancer is the leading cause of cancer-associated mortality worldwide. Smoking is one of the most significant etiological contributors to lung cancer development. However, the molecular mechanisms underlying smoking-induced induction and progression of lung cancer have remained to be fully elucidated. Furthermore, long non-coding RNAs (lncRNAs) are increasingly recognized to have important roles in diverse biological processes. The present study focused on identifying differentially expressed mRNAs, lncRNAs and micro (mi)RNAs in smoking-associated lung cancer. Smoking-associated co-expression networks and protein-protein interaction (PPI) networks were constructed to identify hub lncRNAs and genes in smoking-associated lung cancer. Furthermore, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses of differentially expressed lncRNAs were performed. A total of 314 mRNAs, 24 lncRNAs and 4 miRNAs were identified to be deregulated in smoking-associated lung cancer. PPI network analysis identified 20 hub genes in smoking-associated lung cancer, including dynein axonemal heavy chain 7, dynein cytoplasmic 2 heavy chain 1, WD repeat domain 78, collagen type III α 1 chain (COL3A1), COL1A1 and COL1A2. Furthermore, co-expression network analysis indicated that relaxin family peptide receptor 1, receptor activity modifying protein 2-antisense RNA 1, long intergenic non-protein coding RNA 312 (LINC00312) and LINC00472 were key lncRNAs in smoking-associated lung cancer. A bioinformatics analysis indicated these smoking-associated lncRNAs have a role in various processes and pathways, including cell proliferation and the cyclic guanosine monophosphate cGMP)/protein kinase cGMP-dependent 1 signaling pathway. Of note, these hub genes and lncRNAs were identified to be associated with the prognosis of lung cancer patients. In conclusion, the present study provides useful information for further exploring the diagnostic and prognostic value of the potential candidate biomarkers, as well as their utility as drug targets for smoking-associated lung cancer.

Keywords: co-expression networks; long non-coding RNA; lung cancer; miRNAs; protein-protein interaction analysis; smoking.

Figure 1.

Identification of differently expressed genes, lncRNAs and miRNAs in smoking-associated lung cancer. Hierarchical clustering analysis revealed differentially expressed mRNAs between (A) normal and lung cancer samples and (B) lung cancer samples from smokers and never-smokers from the dataset GSE43458. Hierarchical clustering analysis revealed differential lncRNA expression between (C) normal and lung cancer samples and (D) lung cancer samples from smokers and never-smokers from the dataset GSE43458. Hierarchical clustering analysis reveals differential miRNA expression between (E) normal and lung cancer samples and (F) lung cancer samples from smokers and never-smokers from the dataset TCGA LUAD dataset. lncRNA, long non-coding RNA; miRNA, microRNA.

Figure 2.

Protein-protein interaction network construction and analysis. (A and B) Two hub-networks were distinct for the upregulated smoking-associated genes and (C) one hub-network was identified for the downregulated smoking-associated genes. Red nodes, upregulated genes; blue nodes, downregulated genes.

Figure 3.

Functional analysis of deregulated genes in smoking-associated lung cancer. The DepMap analysis indicates that knockout of (A) CTSK, (B) ITGA11, (C) MYBL2, (D) KPNA2, (E) DNAH7, (F) AURKA and (G) TPX2 significantly suppressed (CERES<0) and (H) knockout of DNAH7 significantly promoted (CERES>0) the proliferation of human cancer cells, including lung cancer. CTSK, cathepsin K; ITGA11, integrin subunit α 11; MYBL2, MYB proto-oncogene like 2; KPNA2, karyopherin subunit α 2; DNAH7, dynein axonemal heavy chain 7; AURKA, aurora kinase A; TPX2, TPX2, microtubule nucleation factor.

Figure 4.

Analysis of the influence of deregulated genes in smoking-associated lung cancer on survival of lung cancer patients. Kaplan-Meier analysis of the effect of 16 hub genes, including (A) AURKA, (B) ITGB4, (C) COL1A1, (D) CDC20, (E) MYBL2, (F) POSTN, (G) COL11A1, (H) TPX2, (I) COL3A1, (J) TIMP1, (K) DNALI1, (L) DNAH6, (M) CTSK, (N) DYNC2H1, (O) DNAH12 and (P) WDR78, on the survival of lung cancer patients. AURKA, aurora kinase A; ITGB4, integrin subunit β 4; COL3A1, collagen type III α 1 chain; CDC20, cell division cycle 20; MYBL2, MYB proto-oncogene like 2; POSTN, periostin; TPX2, TPX2, microtubule nucleation factor; TIMP1, tissue inhibitor of metalloproteinases 1; DNAH6, dynein axonemal heavy chain 6; DNALI1, dynein axonemal light intermediate 1; CTSK, cathepsin K; DYNC2H1, dynein cytoplasmic 2 heavy chain 1; WDR78, WD repeat domain 78.

Figure 5.

Co-expression network analysis of lncRNA-mRNA pairs in smoking-associated lung cancer. Red nodes represent lncRNA and blue nodes represent mRNA. lncRNA, long non-coding RNA.

Figure 6.

GO and KEGG analysis of deregulated lncRNAs in smoking- related lung cancer. Deregulated lncRNAs enriched in (A and B) GO terms in the categories (A) biological process and (B) molecular function, and in (C) KEGG pathways. GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; lncRNA, long non-coding RNA; cGMP, cyclic guanosine monophosphate; PKG, protein kinase.

Figure 7.

Key long non-coding RNAs in smoking-associated lung cancer were downregulated. In LUAD, (A) LINC00312, (B) RXFP1, (C) LINC00472 and (D) RAMP2-AS1 were downregulated. Boxplots indicate the minimum, maximum, median and upper and lower quartiles of gene expression levels in each group. The red boxplot indicates gene expression in normal groups, the blue boxplot indicates gene expression in tumor groups. ***P<0.001. LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; TCGA, The Cancer Genome Atlas; RXFP1, relaxin family peptide receptor 1; RAMP2-AS1, receptor activity modifying protein 2-antisense RNA 1; LINC00312, long intergenic non-protein coding RNA 312.

Figure 8.

Survival analysis of deregulated lncRNAs in smoking-associated lung cancer. (A) The Kaplan-Meier curve analysis indicates that higher expression levels of LINC00472 was weakly associated with a longer overall survival time. (B and C) (B) RAMP2-AS1, (C) LINC00312 and (D) RXFP1 were significantly associated with a longer overall survival time. The median expression of LINC00472, RAMP2-AS1, LINC00312 and RXFP1 were selected as a cutoff to stratify patients into high and low expression groups. The HR values with 95% confidence intervals are provided for the respective lncRNAs. RXFP1, relaxin family peptide receptor 1; RAMP2-AS1, receptor activity modifying protein 2-antisense RNA 1; LINC00312, long intergenic non-protein coding RNA 312; HR, hazard ratio; lncRNA, long non-coding RNA.

Figure 9.

Relative expression of key miRNAs in lung cancer. (A-D) Relative expression of (A) hsa-miR-101-1, (B) hsa-miR-3934, (C) hsa-miR-30e and (D) hsa-miR-190 in normal samples, lung cancer samples from non-smokers and smokers. Boxplots indicate the minimum, maximum, median and upper and lower quartiles of gene expression levels in each group. The red boxplot indicates gene expression in normal groups and the blue boxplot indicates gene expression in tumor groups ***P<0.001. miR, microRNA; hsa, Homo sapiens.