Key microRNAs and hub genes associated with poor prognosis in lung adenocarcinoma

Abstract

In the study, we obtained 36 pairs of lung adenocarcinoma (LUAD) tissues and adjacent non-tumorous tissues. Then, we chose a specific hub-target gene of miRNA and used qRT-PCR to evaluate the expression of PECAM1. We found that the expression level of PECAM1 mRNA in LUAD was significantly lower than that in adjacent nontumor tissues (P<0.0001). Univariate and multivariate analyses were conducted on 481 LUAD patients from The Cancer Genome Atlas (TCGA) according to the Cox proportional hazard regression model to evaluate the impact of PECAM1 expression and other clinicopathological factors on survival. The results showed that the low expression of PECAM1 was an important independent predictor of poor overall survival (HR, 0.704; 95% CI, 0.518-0.957; P = 0.025). Based on the Tumor Immune Estimation Resource (TIMER) database, the relationship between PECAM1 expression and B cell, CD8+ T cell, CD4+ T cell, macrophage, neutrophil, and dendritic cell infiltration was weak in LUAD (P<0.01). In particular, a more significant positive correlation between PECAM1 expression and HLA-complex members, CD1C, NRP1, and ITGAX expression in dendritic cell was detected in LUAD. The mechanism which PECAM1 involved in the development of LUAD may be closely related to changes in the immune microenvironment.

Keywords: PECAM1; differentially expressed genes; differentially expressed miRNAs; lung adenocarcinoma; miRNA-mRNA regulatory network.

Figure 1

Volcano plot of gene expression profile data in LUAD and normal samples and the heatmap of the overlapping DEGs. (A) Volcano plot of GSE74190. (B) Volcano plot of GSE31908. (C) Volcano plot of GSE10072. (D) Volcano plot of GSE43458. (E) Venn diagram of the upregulated overlapping DEGs. (F) Venn diagram of the downregulated overlapping DEGs. (G) Heatmap of the overlapping DEGs. Green represents a low fold change (FC) value, and red represents a high fold change FC value. Each column represents one dataset, and each row represents one gene. The number in each rectangle represents the FC in LUAD samples compared with normal samples. The gradual color change from red to green represents the changing process from upregulation to downregulation.

Figure 2

Functional and pathway enrichment analyses of the overlapping DEGs in LUAD. (A) The BP analysis of DEGs. (B) The MF analysis of DEGs. (C) The CC analysis of DEGs. (D) The KEGG pathway analyses of DEGs. The x-axis represents the q value (−log10), and the y-axis represents the GO term. The GO terms were measured by the rich factor, q value, and number of genes enriched. The greater the rich factor, the greater is the degree of enrichment and the greater the P value [0, 1]. The brighter the color of red, the more significant is the term.

Figure 3

Functional and pathway enrichment analyses of the DEMs in LUAD. (A) The BP analysis of DEMs. (B) the MF analysis of DEMs. (C) The CC analysis of DEMs. (D) The biological pathway analysis of DEMs. The upper x-axis represents the P value (−log10), and the lower x-axis represents the percentage of genes (blue). The y-axis represents the GO term. Yellow represents a P = 0.05 as reference, and red represents the specific P value. The longer the rectangular zone, the smaller is the P value.

Figure 4

PPI network analysis of the overlapping DEGs. Red nodes, upregulated genes; green nodes, downregulated genes.

Figure 5

Overall survival analyses of DEMs. KM curves depicting OS for LUAD patients with high and low expression of (A) hsa-miR-126, (B) hsa-miR-218, (C) hsa-miR-30a, (D) hsa-miR-145, (E) hsa-miR-1, (F) hsa-miR-195, (G) hsa-miR-551b, (H) hsa-miR-497, (I) hsa-miR-101, (J) hsa-miR-215, (K) hsa-miR-31, (L) hsa-miR-21, and (M) hsa-miR-198a.

Figure 6

Prognostic value of eight DEGs in LUAD patients. Prognostic value of (A) SPP1 (log-rank P = 0.0015), (B) COL3A1 (log-rank P = 0.0018), (C) OL1A1 (log-rank P = 4e-08), (D) VWF (log-rank P = 0.045), (E) CDH5 (log-rank P = 0.0034), (F) TEK (log-rank P = 8.9e-10), (G) PECAM1 (log-rank P = 0.0036), and (H) EDN1 (log-rank P = 0.018).

Figure 7

Expression levels of eight hub genes in human lung adenocarcinoma. (A) SPP1; (B) COL3A1; (C) COL1A1; (D) VWF; (E) CDH5; (F) TEK; (G) PECAM1; and (H) EDN1. The gray and red boxes represent normal and cancer tissues, respectively. The expression data are first log₂(TPM+1) transformed for differential analysis, and the log₂FC is defined as median (Tumor, T)-median (Normal, N). *P < 0.05, T vs N. Abbreviations: FC, fold change; TPM, transcript per kilobase million.

Figure 8

Validation of eight hub genes using The Human Protein Atlas database. Expression of (A) SPP1, (B) COL3A1, (C) COL1A1, (D) VWF, (E) CDH5, (F) TEK, (G) PECAM1, and (H) EDN1.

Figure 9

Correlation analysis of upregulated hub genes (SPP1, COL3A1, and COL1A1) and downregulated hub genes (PECAM1, VWF, CDH5, and TEK) in LUAD. (A) SPP1 and COL1A1, (B) SPP1 and COL3A1, (C) COL1A1 and COL3A1, (D) PECAM1 and VWF, (E) PECAM1 and CDH5, (F) PECAM1 and TEK, (G) VWF and CDH5, (H) VWF and TEK, and (I) CDH5 and TEK. TPM, transcript per million. The x-axis represents the TPM of the hub gene MYC (log2). The expression levels of upregulated hub genes (SPP1, COL3A1, and COL1A1) were positively correlated with each other. The expression levels of downregulated hub genes (PECAM1, VWF, CDH5, and TEK) were positively correlated with each other.

Figure 10

ROC curve analysis presenting the sensitivity and specificity of hub genes in LUAD diagnosis. (A) ROC curves of SPP1, (B) COL3A1, (C) COL1A1, (D) VWF, (E) CDH5, (F) TEK, (G) PECAM1, and (H) END1.

Figure 11

Regulatory network of the predicted genes and their target miRNAs. Potential target genes of the significant miRNAs predicted by miRecords.

Figure 12

Comparison of PECAM1 mRNA levels in normal lung tissues and LUAD tissues across four analyses of LUAD. (A–D) Each study showed that the PECAM1 mRNA expression levels in normal lung tissues were significantly higher than those in LUAD tissues. (E) qRT-PCR analysis of PECAM1 mRNA expression in 47 pairs of LUAD tissues and adjacent nontumor tissues. (F) Kaplan-Meier curve of the relationship between PECAM1 mRNA expression and the prognosis of LUAD patients based on the TCGA database.

Figure 13

Correlation of PECAM1 expression with immune cell infiltration levels in LUAD. Tumor-infiltrating immune cells included B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and DCs. Gene expression levels against tumor purity are displayed in the left-most panel.

Figure 14

Correlation of PECAM1 expression with gene markers of tumor-infiltrating immune cells in LUAD. (A) Correlation with gene markers of B cells in LUAD. (B) Correlation with gene markers of CD8+ T cells in LUAD. (C) Correlation with gene markers of CD4+ T cells in LUAD. (D) Correlation with gene markers of neutrophils in LUAD. (E) Correlation with gene markers of macrophages in LUAD. (F) Correlation with gene markers of DCs in LUAD.