Analysis and identification of mRNAsi‑related expression signatures via RNA sequencing in lung cancer

Abstract

High stemness index scores are associated with poor survival in patients with lung cancer. Studies on the mRNA expression-based stemness index (mRNAsi) are typically conducted using tumor tissues; however, mRNAsi-related expression signatures based on cell-free RNA (cfRNA) are yet to be comprehensively investigated. The present study aimed to elucidate the gene expression profiles of tumor stemness in lung cancer tissues and corresponding cfRNAs in blood, and to assess their links with immune infiltration. Tumor tissue, paracancerous tissue, peripheral blood and lymph node samples were collected from patients with stage I-III non-small cell lung cancer and RNA sequencing was performed. The TCGAbiolinks package was used to calculate the mRNAsi for each of these four types of sample. Weighted gene co-expression network analysis and differentially expressed gene analyses were performed to investigate mRNAsi-related genes, and pathway enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology-based annotation system. In addition, the STAR-Fusion tool was used to detect fusion variants, and CIBERSORT was used to analyze the correlations of stemness signatures in tissues and blood with immune cell infiltration. The mRNAsi values in peripheral blood and lymph nodes were found to be higher than those in cancer tissues. 'Hematopoietic cell lineage' was the only KEGG pathway enriched in mRNAsi-related genes in both lung cancer tissues and peripheral blood. In addition, the protein tyrosine phosphatase receptor type C associated protein gene was the only gene commonly associated with the mRNAsi in these two types of sample. The expression of mRNAsi-related genes was increased in the dendritic and Treg cells in tumor tissues, but was elevated in Treg and CD8 cells in the blood. In conclusion, cfRNAs in the blood exhibit unique stemness signatures that have potential for use in the diagnosis of lung cancer.

Keywords: cell-free RNA; diagnosis; immune infiltration; lung cancer; mRNAsi.

Figure 1.

Relationships between mRNAsi values and overall survival in patients with non-small cell lung cancer assessed using K-M analysis. K-M comparison of high and low mRNAsi groups in (A) peripheral blood and (B) tumor tissues. mRNAsi, mRNA expression-based stemness index; K-M, Kaplan-Meier; P-high, high peripheral blood mRNAsi; P-low, low peripheral blood mRNAsi; T-high, high tumor tissue mRNAsi; T-low, low tumor tissue mRNAsi.

Figure 2.

Comparison of mRNAsi among different matched patient samples. All groups were compared by the Friedman test, and comparisons between two groups were performed using Wilcoxon signed-rank tests with Bonferroni correction. **P<0.01, ***P<0.001. N, normal paracancerous tissues; T, tumor tissues; P, peripheral blood; L, lymph node.

Figure 3.

Genes and pathways associated with lung cancer. (A) Pathway enrichment analysis of the genes found to be associated with lung cancer by weighted gene co-expression network analysis. (B) DEGs associated with lung tumorigenesis. Pathways enriched by (C) upregulated DEGs and (D) downregulated DEGs. DEGs, differentially expressed genes; TvsN, tumor tissues vs. normal paracancerous tissues; Up, upregulated genes; Down, downregulated genes; NS, not significant; P-adj, adjusted P-value.

Figure 4.

Genes and pathways associated with the mRNAsi of lung cancer tissues and peripheral blood. (A) WGCNA was used to analyze the genes associated with the mRNAsi in tissues and (B) enriched KEGG pathways were identified. Pathways enriched in (C) upregulated and (D) downregulated DEGs associated with the tissue mRNAsi. (E) WGCNA analysis of the genes associated with the mRNAsi in peripheral blood and (F) enriched KEGG pathways. Pathways enriched in (G) upregulated and (H) downregulated DEGs associated with the mRNAsi of peripheral blood. mRNAsi, mRNA expression-based stemness index; WGCNA, weighted gene co-expression network analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; DEGs, differentially expressed genes.

Figure 5.

Immune cells associated with the stemness of lung cancer tissue and cfRNA. (A) Analysis of the infiltration of various immune cells in tissues. (B) Correlation between the mRNAsi-related genes of tissues and immune cell infiltration. (C) Correlation between cfRNA stemness index-related genes and immune cell infiltration. Correlation between immune cells and the (D) mRNAsi of tumor tissues and (E) cfRNA stemness index. cfRNA, cell-free DNA; mRNAsi, mRNA expression-based stemness index.