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Cited by 567Extracellular vesicles in diseaseMicroRNA in disease regulationCancer Cells and Metastasis

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Hypoxic BMSC-derived exosomal miRNAs promote metastasis of lung cancer cells via STAT3-induced EMT

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Zhang, Xina, Sai, Buqing, Wang, Fan, Wang, Lujuan, Wang, Yuhui, Zheng, Leliang, Li, Guiyuan, Tang, Jingqun, Xiang, Juanjuan·2019·Molecular Cancer
DOI 10.1186/s12943-019-0959-5PMC PMC6417285
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Abstract

BACKGROUND: Metastasis is the main cause of lung cancer mortality. Bone marrow-derived mesenchymal stem cells (BMSCs) are a component of the cancer microenvironment and contribute to cancer progression. Intratumoral hypoxia affects both cancer and stromal cells. Exosomes are recognized as mediators of intercellular communication. Here, we aim to further elucidate the communication between BMSC-derived exosomes and cancer cells in the hypoxic niche. METHODS: Exosomal miRNA profiling was performed using a microRNA array. Lung cancer cells and an in vivo mouse syngeneic tumor model were used to evaluate the effects of select exosomal microRNAs. Hypoxic BMSC-derived plasma exosomal miRNAs were assessed for their capacity to discriminate between cancer patients and non-cancerous controls and between cancer patients with or without metastasis. RESULTS: We demonstrate that exosomes derived from hypoxic BMSCs are taken by neighboring cancer cells and promote cancer cell invasion and EMT. Exosome-mediated transfer of select microRNAs, including miR-193a-3p, miR-210-3p and miR-5100, from BMSCs to epithelial cancer cells activates STAT3 signaling and increases the expression of mesenchymal related molecules. The diagnostic accuracy of individual microRNA showed that plasma exosomal miR-193a-3p can discriminate cancer patients from non-cancerous controls. A panel of these three plasma exosomal microRNAs showed a better diagnostic accuracy to discriminate lung cancer patients with or without metastasis than individual exosomal microRNA. CONCLUSIONS: Exosome-mediated transfer of miR-193a-3p, miR-210-3p and miR-5100, could promote invasion of lung cancer cells by activating STAT3 signalling-induced EMT. These exosomal miRNAs may be promising noninvasive biomarkers for cancer progression.

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Fig. 1 Identifying exosomal miRNAs secreted from hypoxic BMSCs that improve the invasion of lung cancer Cells. LLC cells that were stably transfected with the firefly luciferase gene (luci-LLC) were subcutaneously injected with or without BMSCs into C57BL/6 mice. 28 days after treatment, the bioluminescence imaging system was used to monitor the mobility and growth of LLC cells in vivo. a Bioluminescent images were shown. The activities of luciferase were shown in histogram. b Representative images of lung metastatic nodules from mice. c Representative images of Hematoxylin and Eosin (HE) staining of lungs section. HE staining was performed to detect the metastatic nodules in the lung. d Representative upregulated or downregulated plasma exosomal microRNAs from mice that received co-injection of BMSCs and LLC cells in contrast to mice that received injection of LLC cells alone. e The BMSCs were cultured in a hypoxic chamber for 3 days. The exosomes were collected and isolated from the cell culture medium. The exosomal miRNA expression profile was examined.Hierarchical clustering analysis of exosomal microRNA expression. Signals were normalized using Gene Spring GX software 11.0. Clustering was performed on differentially expressed exosomal microRNAs between hypoxic BMSC-secreted exosomes and normoxic BMSC-secreted exosome. Columns represent individual samples and rows represent each exosomal microRNA. Red and green in cells reflect high and low expression levels, respectively, as indicated in the scale bar (log2-transformed scale). f Representative upregulated or downregulated microRNAs from hypoxic BMSC- secreted exosomes compared to normoxic BMSC-secreted exosomes. g Venn diagram showed the overlap of shared exosomal miRNAs in vitro and in vivo. h Expression of selected exosomal miRNAs was verified by quantitative real-time PCR. Experiments were performed in triplicate. Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 1 Identifying exosomal miRNAs secreted from hypoxic BMSCs that improve the invasion of lung cancer Cells. LLC cells that were stably transfected with the firefly luciferase gene (luci-LLC) were subcutaneously injected with or without BMSCs into C57BL/6 mice. 28 days after treatment, the bioluminescence imaging system was used to monitor the mobility and growth of LLC cells in vivo. a Bioluminescent images were shown. The activities of luciferase were shown in histogram. b Representative images of lung metastatic nodules from mice. c Representative images of Hematoxylin and Eosin (HE) staining of lungs section. HE staining was performed to detect the metastatic nodules in the lung. d Representative upregulated or downregulated plasma exosomal microRNAs from mice that received co-injection of BMSCs and LLC cells in contrast to mice that received injection of LLC cells alone. e The BMSCs were cultured in a hypoxic chamber for 3 days. The exosomes were collected and isolated from the cell culture medium. The exosomal miRNA expression profile was examined.Hierarchical clustering analysis of exosomal microRNA expression. Signals were normalized using Gene Spring GX software 11.0. Clustering was performed on differentially expressed exosomal microRNAs between hypoxic BMSC-secreted exosomes and normoxic BMSC-secreted exosome. Columns represent individual samples and rows represent each exosomal microRNA. Red and green in cells reflect high and low expression levels, respectively, as indicated in the scale bar (log2-transformed scale). f Representative upregulated or downregulated microRNAs from hypoxic BMSC- secreted exosomes compared to normoxic BMSC-secreted exosomes. g Venn diagram showed the overlap of shared exosomal miRNAs in vitro and in vivo. h Expression of selected exosomal miRNAs was verified by quantitative real-time PCR. Experiments were performed in triplicate. Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001CC BY · PubMed Central
Fig. 2 Hypoxic-BMSC-derived exosomes promote metastasis of lung cancer cells via EMT. a Cell migration and invasion were measured by transwell assays. A549 cells and LLC cells were treated with hypoxic BMSC-secreted or normoxic BMSC-secreted exosomes for 48 h. Cells that migrated or invaded to the bottom surface were stained with crystal violet and observed by light microscopy (magnification, 100×). b The numbers of migrating cells or invading cells were counted from six fields of view in each group. Data were presented as the mean ± SD, and analyzed with Student’s t-test. *P < 0.05; ** < 0.01; *** < 0.001. c Epithelial and mesenchymal markers in exosome-treated lung cancer cells were measured by quantitative real-time PCR. Experiments were performed in triplicate. *P < 0.05; ** < 0.01. d Bioluminescent images were shown. Timeline of experimental protocol was shown above. C57BL/6 mice were subcutaneously injected with LLC-luciferase cells. When the mean tumor volume reached 100 mm3, the mice were intratumorally injected with exosomes released from hypoxic BMSCs or normoxic BMSCs. Activities of luciferase were shown in histogram. e Representative images of metastatic tumor nodules formed in the lungs
Fig. 2 Hypoxic-BMSC-derived exosomes promote metastasis of lung cancer cells via EMT. a Cell migration and invasion were measured by transwell assays. A549 cells and LLC cells were treated with hypoxic BMSC-secreted or normoxic BMSC-secreted exosomes for 48 h. Cells that migrated or invaded to the bottom surface were stained with crystal violet and observed by light microscopy (magnification, 100×). b The numbers of migrating cells or invading cells were counted from six fields of view in each group. Data were presented as the mean ± SD, and analyzed with Student’s t-test. *P < 0.05; ** < 0.01; *** < 0.001. c Epithelial and mesenchymal markers in exosome-treated lung cancer cells were measured by quantitative real-time PCR. Experiments were performed in triplicate. *P < 0.05; ** < 0.01. d Bioluminescent images were shown. Timeline of experimental protocol was shown above. C57BL/6 mice were subcutaneously injected with LLC-luciferase cells. When the mean tumor volume reached 100 mm3, the mice were intratumorally injected with exosomes released from hypoxic BMSCs or normoxic BMSCs. Activities of luciferase were shown in histogram. e Representative images of metastatic tumor nodules formed in the lungsCC BY · PubMed Central
Fig. 3 Exosome-mediated transfer of miR-193a, miR-210-3p and miR-5100 from BMSCs promotes invasion of cancer cells. a Cell invasion were measured by transwell assays. Cells were transfected with mimics of miR-193a-3p, miR-210-3p and miR-5100. Cells that invaded to the bottom surface were stained with crystal violet and observed by light microscopy (magnification, 100×). The numbers of invading cells were counted from six fields of view in each group. Data were presented as the mean ± SD, and analyzed with Student’s t-test. *P < 0.05; ** < 0.01; *** < 0.001. b Cell invasion were measured by transwell assays. LLC cells were treated with hypoxic BMSC-secreted exosomes followed by transfection with inhibitors of miR-193a-3p, miR-210-3p and miR-5100. Cells that invaded to the bottom surface were stained with crystal violet and observed by light microscopy (magnification, 100×). The numbers of migrating cells or invading cells were counted from six fields of view in each group. Data were presented as the mean ± SD, and analyzed with Student’s t-test. *P < 0.05; ** < 0.01; *** < 0.001. c The expression of epithelial and mesenchymal markers in LLC cells measured by quantitative real-time PCR. LLC cells were treated with hypoxic BMSC-secreted exosomes followed by transfection with inhibitors of miR-193a-3p, miR-210-3p and miR-5100. Experiments were performed in triplicate. Data were presented as the mean ± SD, and analyzed with Student’s t-test. *P < 0.05; ** < 0.01; *** < 0.001
Fig. 3 Exosome-mediated transfer of miR-193a, miR-210-3p and miR-5100 from BMSCs promotes invasion of cancer cells. a Cell invasion were measured by transwell assays. Cells were transfected with mimics of miR-193a-3p, miR-210-3p and miR-5100. Cells that invaded to the bottom surface were stained with crystal violet and observed by light microscopy (magnification, 100×). The numbers of invading cells were counted from six fields of view in each group. Data were presented as the mean ± SD, and analyzed with Student’s t-test. *P < 0.05; ** < 0.01; *** < 0.001. b Cell invasion were measured by transwell assays. LLC cells were treated with hypoxic BMSC-secreted exosomes followed by transfection with inhibitors of miR-193a-3p, miR-210-3p and miR-5100. Cells that invaded to the bottom surface were stained with crystal violet and observed by light microscopy (magnification, 100×). The numbers of migrating cells or invading cells were counted from six fields of view in each group. Data were presented as the mean ± SD, and analyzed with Student’s t-test. *P < 0.05; ** < 0.01; *** < 0.001. c The expression of epithelial and mesenchymal markers in LLC cells measured by quantitative real-time PCR. LLC cells were treated with hypoxic BMSC-secreted exosomes followed by transfection with inhibitors of miR-193a-3p, miR-210-3p and miR-5100. Experiments were performed in triplicate. Data were presented as the mean ± SD, and analyzed with Student’s t-test. *P < 0.05; ** < 0.01; *** < 0.001CC BY · PubMed Central
Fig. 4 The exosomal miRNAs were transferred from BMSCs to the cancer cells. a Fluorescently labeled exosomes entered into LLC cells. Representative images were filmed after cells were fixed and stained (magnification, 400×). b The expression of miR-193a-3p, miR-210-3p and miR-5100 in cancer cells after treatment measured by Quantitative real-time PCR. LLC cells were treated with normoxic or hypoxic BMSC-secreted exosomes. Experiments were performed in triplicate.*P < 0.05. c The expression of exosomal miR-193a-3p, miR-210-3p and miR-5100 measured by Quantitative real-time PCR. Exosomal microRNAs were isolated from hypoxic BMSCs that treated with GW4869. Experiments were performed in triplicate Student’s t-test. *P < 0.05; ** < 0.01; *** < 0.001. d LLC cells were isolated by flow cytometry cell sorting. Timeline of experimental protocol was shown above. C57BL/6 mice were subcutaneously injected with LLC-RFP with or without BMSCs. When the mean tumor volume reached 150–200 mm3, the red fluorescent protein positive-LLC cells were collected from the tumor sites by flow cytometry cell sorting. e The levels of miR-193a-3p, miR-210-3p and miR-1500 in sorted LLC cells measured by quantitative real-time PCR. Experiments were performed in triplicate. Student’s t-test. *P < 0.05; ** < 0.01; *** < 0.001
Fig. 4 The exosomal miRNAs were transferred from BMSCs to the cancer cells. a Fluorescently labeled exosomes entered into LLC cells. Representative images were filmed after cells were fixed and stained (magnification, 400×). b The expression of miR-193a-3p, miR-210-3p and miR-5100 in cancer cells after treatment measured by Quantitative real-time PCR. LLC cells were treated with normoxic or hypoxic BMSC-secreted exosomes. Experiments were performed in triplicate.*P < 0.05. c The expression of exosomal miR-193a-3p, miR-210-3p and miR-5100 measured by Quantitative real-time PCR. Exosomal microRNAs were isolated from hypoxic BMSCs that treated with GW4869. Experiments were performed in triplicate Student’s t-test. *P < 0.05; ** < 0.01; *** < 0.001. d LLC cells were isolated by flow cytometry cell sorting. Timeline of experimental protocol was shown above. C57BL/6 mice were subcutaneously injected with LLC-RFP with or without BMSCs. When the mean tumor volume reached 150–200 mm3, the red fluorescent protein positive-LLC cells were collected from the tumor sites by flow cytometry cell sorting. e The levels of miR-193a-3p, miR-210-3p and miR-1500 in sorted LLC cells measured by quantitative real-time PCR. Experiments were performed in triplicate. Student’s t-test. *P < 0.05; ** < 0.01; *** < 0.001CC BY · PubMed Central
Fig. 5 Exosomal miRNAs from BMSCs promote metastasis by STAT3 driven EMT. C57BL/6 mice were subcutaneously injected with LLC-RFP with or without BMSCs. The red fluorescent protein positive LLC cells were collected from the tumor sites by flow cytometry cell sorting and subjected to RNA sequencing analysis. Timeline of experimental protocol was shown. a The upregulated genes were enriched in the JAK-STAT pathway. Clustering was performed on differentially expressed mRNAs between LLC cells collected from mice that received co-injection of BMSCs and LLC or LLC injection alone. Columns represent individual samples and rows represent each gene. Red and green reflect high and low expression levels, respectively. FPKM for STAT3 transcripts obtained by RNA-Seq was shown in histogram. b Protein expression of STAT3, p-STAT3 were measured by Western blot analysis. Cells were treated with normoxic BMSC-secreted exosomes or hypoxic BMSC-secreted exosomes. β-actin was used as the internal control. c Cell invasion was measured by transwell assay. The increased invasion capability induced by hypoxic BMSC-secreted exosomes was reversed by stat3 inhibitor stattics. Magnification, 100×. d Expression of Snail and Vimentin was measured by quantitative real-time PCR. Cells were treated with hypoxic BMSC-secreted exosomes with or without stat3 inhibitor stattics. Experiments were performed in triplicate.*P < 0.05. e Expression of stat3 measured by western blot. The miR-193a-3p inhibitor, miR-210-3p inhibitor and miR-5100 inhibitor could reverse the enhanced expression of phosphorylated STAT3 induced by exosomes released from hypoxia
Fig. 5 Exosomal miRNAs from BMSCs promote metastasis by STAT3 driven EMT. C57BL/6 mice were subcutaneously injected with LLC-RFP with or without BMSCs. The red fluorescent protein positive LLC cells were collected from the tumor sites by flow cytometry cell sorting and subjected to RNA sequencing analysis. Timeline of experimental protocol was shown. a The upregulated genes were enriched in the JAK-STAT pathway. Clustering was performed on differentially expressed mRNAs between LLC cells collected from mice that received co-injection of BMSCs and LLC or LLC injection alone. Columns represent individual samples and rows represent each gene. Red and green reflect high and low expression levels, respectively. FPKM for STAT3 transcripts obtained by RNA-Seq was shown in histogram. b Protein expression of STAT3, p-STAT3 were measured by Western blot analysis. Cells were treated with normoxic BMSC-secreted exosomes or hypoxic BMSC-secreted exosomes. β-actin was used as the internal control. c Cell invasion was measured by transwell assay. The increased invasion capability induced by hypoxic BMSC-secreted exosomes was reversed by stat3 inhibitor stattics. Magnification, 100×. d Expression of Snail and Vimentin was measured by quantitative real-time PCR. Cells were treated with hypoxic BMSC-secreted exosomes with or without stat3 inhibitor stattics. Experiments were performed in triplicate.*P < 0.05. e Expression of stat3 measured by western blot. The miR-193a-3p inhibitor, miR-210-3p inhibitor and miR-5100 inhibitor could reverse the enhanced expression of phosphorylated STAT3 induced by exosomes released from hypoxiaCC BY · PubMed Central
Fig. 6 Exosomal miRNA can be biomarkers for cancer metastasis. Plasma samples were collected from 72 patients with pancreatic cancer, lung cancer or liver cancer and 30 healthy controls. Plasma exosomes were isolated. a Expression of miR-193a-3p and miR-210-3p and miR-5100 in plasma exosomes from the lung cancer patients (n = 41),liver cancer patients(n = 21),pancreas cancer patients(n = 10),and normal healthy control (n = 30). b Expression of miR-193a-3p and miR-210-3p and miR-5100 in plasma exosomes from the metastatic lung cancer patients (n = 21),non-metastatic lung cancer patients (n = 20) and normal samples (n = 30). c ROC analysis of the individual exosomal miRNA and the three exosomal miRNA panel for cancer patients and non-cancerous control. d ROC analysis of the individual exosomal miRNA and the three exosomal miRNAs panel for the metastatic lung cancer patients and non-metastatic lung cancer patients
Fig. 6 Exosomal miRNA can be biomarkers for cancer metastasis. Plasma samples were collected from 72 patients with pancreatic cancer, lung cancer or liver cancer and 30 healthy controls. Plasma exosomes were isolated. a Expression of miR-193a-3p and miR-210-3p and miR-5100 in plasma exosomes from the lung cancer patients (n = 41),liver cancer patients(n = 21),pancreas cancer patients(n = 10),and normal healthy control (n = 30). b Expression of miR-193a-3p and miR-210-3p and miR-5100 in plasma exosomes from the metastatic lung cancer patients (n = 21),non-metastatic lung cancer patients (n = 20) and normal samples (n = 30). c ROC analysis of the individual exosomal miRNA and the three exosomal miRNA panel for cancer patients and non-cancerous control. d ROC analysis of the individual exosomal miRNA and the three exosomal miRNAs panel for the metastatic lung cancer patients and non-metastatic lung cancer patientsCC BY · PubMed Central

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  1. Fang JH, Zhang ZJ, Shang LR, Luo YW, Lin YF, Yuan Y, et al. Hepatoma cell-secreted exosomal microRNA-103 increases vascular permeability and promotes metastasis by targeting junction proteins. Hepatology. 2018;68(4):1459–75. · DOI

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Metadata

Authors
Zhang, X., Sai, B., Wang, F., Wang, L., Wang, Y., Zheng, L., Li, G., Tang, J., & Xiang, J.
Affiliations
Central South University (CN)
Corresponding author
  • Tang, Jingqun
  • Xiang, Juanjuan
ORCID
Venue
Molecular Cancer
Publisher
Springer Science and Business Media LLC
Published
2019-03-13
Volume
18
Issue
1
Pages
40-40
ISSN
1476-4598 (ISSN-L)
DOI
10.1186/s12943-019-0959-5
PMC
PMC6417285
OpenAlex
W2938403043
Cited by
560
References
50
Citations by year
2026: 34 · 2025: 61 · 2024: 96 · 2023: 94 · 2022: 114 · 2021: 81 · 2020: 63 · 2019: 16
FWCI
23.01
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Cancer (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Natural Science Foundation of China (81472695, 81773147)
  • Strategic Priority Research Program of Central South University (ZLXD2017004)
  • National Training and Research Base for Talents of principles of carcinogenesis foundation (111 project: 111-2-12)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Health Sciences › Medicine › Oncology › Cancer Cells and Metastasis
Keywords
lung cancer metastasis, bone marrow-derived mesenchymal stem cells, exosomes, exosomal microRNAs, hypoxia, STAT3 signaling, epithelial-mesenchymal transition, tumor invasion, miR-193a-3p, miR-210-3p, tumor microenvironment, exosome-mediated intercellular communication
MeSH terms
Animals, Hypoxia, Cell Movement, Female, Follow-Up Studies, Humans, Lung Neoplasms, Male, Mice, Inbred C57BL, Mice, Nude, Middle Aged, Prognosis, Tumor Cells, Cultured, Biomarkers, Tumor, Signal Transduction, Gene Expression Regulation, Neoplastic, Survival Rate, Case-Control Studies, Apoptosis, Xenograft Model Antitumor Assays, MicroRNAs, Cell Proliferation, STAT3 Transcription Factor, Mice, Exosomes, Epithelial-Mesenchymal Transition, Mesenchymal Stem Cells

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