Roles of hypoxic environment and M2 macrophage-derived extracellular vesicles on the progression of non-small cell lung cancer

Abstract

Background: Hypoxia contributes to the development of invasive and metastatic cancer cells, and is detrimental to cancer treatment. This study aimed to explore the molecular mechanisms by which hypoxic microenvironments affect hypoxic non-small cell lung cancer (NSCLC) development and the effects of M2 macrophage-derived extracellular vesicles (EVs) on NSCLC cells.

Methods: A549 cells were cultured in an anoxic incubator for 48 h to construct hypoxic A549 cells, and then normal and hypoxic A549 cells were harvested for RNA sequencing. Next, THP-1 cells were used to induce M2 macrophages, and EVs were isolated from THP-1 cells and M2 macrophages. Cell counting kit-8 and transwell assays were used to determine the viability and migration of hypoxic A549 cells, respectively.

Results: After sequencing, 2426 DElncRNAs and 501 DEmiRNAs were identified in normal A549 cells and hypoxic A549 cells. These DElncRNAs and DEmiRNAs were significantly enriched in "Wnt signaling pathway," "Hippo signaling pathway," "Rap1 signaling pathway," "calcium signaling pathway," "mTOR signaling pathway," and "TNF signaling pathway." Subsequently, ceRNA networks consisting of 4 lncRNA NDRG1 transcripts, 16 miRNAs and 221 target mRNAs were built, and the genes in the ceRNA networks were significantly associated with "Hippo signaling pathway" and "HIF-1 signaling pathway." EVs were successfully extracted from THP-1 cells and M2 macrophages, and M2 macrophage-derived EVs significantly enhanced the viability and migration of hypoxic A549 cells. Finally, M2 macrophage-derived EVs further upregulated the expression of NDRG1-009, NDRG1-006, VEGFA, and EGLN3, while downregulating miR-34c-5p, miR-346, and miR-205-5p in hypoxic A549 cells.

Conclusions: M2 macrophage-derived EVs may worsen the progression of NSCLC in a hypoxic microenvironment by regulating the NDRG1-009-miR-34c-5p-VEGFA, NDRG1-006-miR-346-EGLN3, NDRG1-009-miR-205-5p-VEGFA, and Hippo/HIF-1 signaling pathways.

Keywords: Extracellular vesicles; Hypoxia; M2 macrophages; Non-small cell lung cancer.

Fig. 1

Identification of differential expressed long non-coding RNAs (DElncRNAs) and their functional analyses. (A) Volcano plots of DElncRNAs based on the thresholds of |log₂Fold change (FC)| > 1 and adjusted P value (padj) < 0.05. Red and blue dots respectively represented the up-regulated and down-regulated DElncRNAs. (B) A bidirectional hierarchical clustering heatmap of the identified DElncRNAs. (C) Top10 gene ontology (GO) terms of biological process (BP), cellular component (CC) and molecular function (MF) of these identified DElncRNAs. (D) Significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the identified DElncRNAs.

Fig. 2

Analysis of differential expressed microRNAs (DEmiRNAs). (A) Volcano plots of DEmiRNAs based on the thresholds of |log₂FC| > 1 and padj < 0.05. Red and blue dots respectively denoted the up-regulated and down-regulated DEmiRNAs. (B) A bidirectional hierarchical clustering heatmap of the screened DEmiRNAs. (C) GO terms of these screened DEmiRNAs. (D) Significantly enriched KEGG pathways of the screened DEmiRNAs.

Fig. 3

Construction of competitive endogenous RNA (ceRNA) neworks and functional analyses of these genes in the ceRNA networks. (A) CeRNA networks about lncRNA NDRG1. Squares, rhombuses, and circles represented lncRNAs, miRNAs, and mRNAs, respectively. (B) GO terms analysis of the genes in the ceRNA networks. (C) Significantly enriched KEGG pathway of the genes in the ceRNA networks

Fig. 4

Validation of selected three ceRNA pairs and induction of M2 macrophages using real-time quantitative PCR. (A) The expression levels of lncRNA NDRG1-009-miR-34c-5p-VEGFA. (B) The expression levels of lncRNA NDRG1-006-miR-346-EGLN3. (C) The expression levels of lncRNA NDRG1-009-miR-205-5p-VEGFA. *: P < 0.05, compared with the blank group. (D) The relative expression of M2 macrophages-related markers TNF-β, IL-10, Arg-1, YM1, CD206, and CD163 in the THP-1 cells and M2 macrophages. *: P < 0.05, compared with the THP-1 cells

Fig. 5

Characterization of the extracellular vesicles (EVs) from THP-1 cells and M2 macrophages. (A) The morphology of EVs isolated from THP-1 cells and M2 macrophages was observed by a transmission electron microscopy. (B) A Nanosight NS300 particle size analyser was used to measure the particle size of THP-1 cells-derived EVs and M2 macrophages-derived EVs. (C) Western blot was used to detect the expression of the EVs-specific markers (CD9, HSP70 and TSG101). (D) Cellular uptake of PKH67-labeled exosomes (green fluorescence) and hypoxic A549 cells after co-cultured for 48 h. hy-A549: hypoxic A549 cells; THP-1-EVs: THP-1 cells-derived EVs; M2-EEVs: M2 macrophages-derived EVs.

Fig. 6

Effects of M2 macrophages-derived EVs on the growth of hypoxic A549 cells. (A) Cell viability of hypoxic A549 cells treated with different concentrations of different EVs was determined by cell counting kit-8. (B) Cell migration of hypoxic A549 cells treated with different EVs was measured using Transwell. *: P < 0.05, compared with the hypoxic A549 cells. ^#: P < 0.05, compared with the hypoxic A549 cells treated with THP-1 cells-derived EVs. hy-A549: hypoxic A549 cells; THP-1-EVs: THP-1 cells-derived EVs; M2-EVs: M2 macrophages-derived EVs.

Fig. 7

Effects of M2 macrophages-derived EVs on the expression of the related lncRNAs, miRNAs and mRNAs in the hypoxic A549 cells by RT-qPCR. (A) The mRNA expression of lncRNA NDRG1-009 and NDRG1-006. (B) The mRNA expression of miR-34c-5p, miR-346 and miR-205-5p. (C) The mRNA expression of VEGFA and EGLN3. *: P < 0.05, compared with the hypoxic A549 cells. ^#: P < 0.05, compared with the hypoxic A549 cells treated with THP-1 cells-derived EVs. hy-A549: hypoxic A549 cells; THP-1-EVs: THP-1 cells-derived EVs; M2-EVs: M2 macrophages-derived EVs.