Hypoxic tumor-derived exosomal miR-31-5p promotes lung adenocarcinoma metastasis by negatively regulating SATB2-reversed EMT and activating MEK/ERK signaling

Abstract

Background: Exosomes have emerged as critical mediators of intercellular communication. Hypoxia is widely recognized as a key regulator of tumor aggressiveness, and significantly affects exosome release by tumor cells. However, the effects of exosomes derived from hypoxic lung adenocarcinoma (LUAD) cells are poorly understood.

Methods: Samples of miRNA isolated from hypoxic LUAD cell-derived exosomes (HExo) and normoxic LUAD cell-derived exosomes (NExo) were sequenced to identify miRNAs that might mediate tumor progression. Exosomal miRNA was co-cultured with LUAD cells to assess its biological effects on cell migration and metastasis both in vitro and in vivo. The cellular target of exosomal miRNA was confirmed by dual-luciferase assays. Western blot studies showed that exosomal miRNA regulated the related pathway. The availability of circulating exosomal miRNA derived from plasma was also evaluated.

Results: We found that HExo could significantly enhance the migration and invasion of normoxic LUAD cells. MiRNA sequencing results suggested that miR-31-5p was largely internalized within HExo and could be taken up by normoxic LUAD cells. Exosomal miR-31-5p was found to directly target Special AT-Rich Sequence-Binding Protein 2 (SATB2)-revered epithelial mesenchymal transition and significantly increase activation of MEK/ERK signaling, thereby contributing to tumor progression both in vitro and in vivo. Furthermore, higher levels of circulating exosomal miR-31-5p were detected in LUAD patients, especially in patients with metastatic disease.

Conclusions: Our findings demonstrate that exosomal miR-31-5p exerts a crucial role in LUAD progression, and could serve as a diagnostic biomarker for LUAD.

Keywords: Lung adenocarcinoma; SATB2; exosome; hypoxia; miR-31-5p.

Fig. 1

Characterization of exosomes derived from LUAD cells. a Expression of HIF-1α in A549 and H1299 cells under hypoxic and normoxic conditions. b Representative TEM picture of exosomes derived from LUAD cells. c NTA were detected the concentration and size distribution of HExo. d Comparison of exosomes concentration between HExo and NExo. e Western blot analysis for presence of exosomes markers, CD63, TSG101, Flotillin and absence of negative control, Calnexin. Data are presented as the mean ± SD of three independent experiments (**, P<0.01)

Fig. 2

Effects of HExo on LUAD. a Internalization of PKH67-labeled exosomes by LUAD cells. Scale bar, 20 μm. b Western blot analysis for E-cadherin and Vimentin in the control (PBS), NExo and HExo groups. (c-d) Scratch assays and migration assays were assessed in the control (PBS), NExo and HExo groups, respectively. Scale bar, 20 μm. (e) Invasion assays were analyzed in the control (PBS), NExo and HExo groups. Scale bar, 20 μm. Data are represented as the mean ± SD from three independent experiments (*, P<0.05; **, P<0.01; ***, P<0.001)

Fig. 3

miR-31-5p was encapsuled in HExo. a Expression of exosomal miR-31-5p in HExo and NExo of four LUAD cell lines. b Expression of exosomal miR-31-5p in nontreated, RNase A-treated and RNase A + Triton X-100-treated groups. c Expression of exosomal miR-31-5p in NExo, HExo and HExo + GW4869 groups. Data are represented as the mean ± SD from three independent experiments (**, P<0.01)

Fig. 4

Exosomal miR-31-5p directly targeted SATB2. a-b Luciferase activities of WT or mut 3’UTR in A549 cells after transfection of miR-31-5p mimic or HExo. c Expression of SATB2 after transfection miR-31-5p mimic under hypoxic or normoxic conditions. d Expression of SATB2 after treatment of HExo. Scratch assays (e) and Transwell invasion assays (f) results after transfection of si-SATB2 and/or miR-31-5p inhibitor in A549 cell line. Data are represented as the mean ± SD from three independent experiments (*, P<0.05; **, P<0.01; ***, P<0.001)

Fig. 5

Exosomal miR-31-5p promoted metastasis of LUAD cells in vivo. a Representative images of lung metastasis in nude mice after tail intravenous injection of A549 cells treated with HExo or miR-31-5p inhibitor (31-inhibitor) or treated with HExo and miR-31-5p inhibitor (HExo + 31-inhibitor). Scale bar, Morphology 500 mm; HE 50 μm. b Numbers of metastatic lung nodules in each group. c Overall survival times of each group after tail intravenous injection of A549 cells. d Representative pictures of E-cadherin, Vimentin and SATB2 expression in lung metastatic nodule by immunohistochemistry staining. Scale bar, 50 μm

Fig. 6

Exosomal miR-31-5p activated MEK/ERK signaling pathway. a Phosphorylation of MEK and ERK were determined after treatment of HExo. b EMT markers, E-cadherin and vimentin, phosphorylation of MEK and ERK were determined after treatment of HExo and/or miR-31-5p inhibitor. Scratch assays (c) and Transwell invasion assays (d) results after treatment of HExo and/or miR-31-5p inhibitor. Scale bar, 20 μm. Data are represented as the mean ± SD from three independent experiments (**, P<0.01; ***, P<0.001)

Fig. 7

Exosomal miR-31-5p might act as a diagnostic biomarker for LUAD. a A representative TEM image of plasma-derived exosomes from lung adenocarcinoma patients. b Expression of exosomal miR-31-5p in plasma-derived from lung adenocarcinoma patients and healthy controls. c Expression of exosomal miR-31-5p from plasma in metastatic patients and non-metastatic ones. d ROC curve for plasma-derived exosomal miR-31-5p. e Schematic diagram of hypoxic exosomes containing miR-31-5p transferred to normoxic cells contributing to LUAD invasion and migration by negatively regulating SATB2-reversed EMT and activating MEK/ERK signaling