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Cited by 245Extracellular vesicles in diseaseMicroRNA in disease regulationNanoparticle-Based Drug Delivery

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Exosomes derived from gemcitabine-resistant cells transfer malignant phenotypic traits via delivery of miRNA-222-3p

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Wei, Feng, Ma, Chengyuan, Zhou, Tong, Dong, Xuechao, Luo, Qinghua, Geng, Li, Ding, Lijuan, Zhang, Yandong, Zhang, Li, Li, Nan, Li, Yang, Liu, Yan·2017·Molecular Cancer
DOI 10.1186/s12943-017-0694-8PMC PMC5526308
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Abstract

BACKGROUND: Although gemcitabine-based chemotherapy has been established as a core multimodal therapy for non-small cell lung cancer (NSCLC) treatment, its clinical efficacy remains limited by the development of acquired resistance following tumor metastasis and relapse. In this study, we investigated how gemcitabine-resistant (GR) cells contribute to the development of NSCLC tumor malignancy via exosome-mediated transfer of microRNAs. METHODS: We first studied the mechanism of exosome internalization via PKH-67 staining and an immunofluorescence assay, then confirmed our finding by transmission electron microscopy and western blot analysis. Candidate miRNAs were identified through microarray analysis. Thereafter, RT-PCR, MTS, Transwell and soft agar assays were performed to assess the role of exosomic miR-222-3p in vitro. A 3' untranslated region reporter assay was applied to identify the target of miR-222-3p. A lung metastasis mouse model was constructed to evaluate tumor growth and metastasis in vivo. Finally, clinical samples were used for correlation analysis between exosomic miR-222-3p levels and patients' response to gemcitabine. RESULTS: A549-GR-derived exosomes were internalized by receipt cells via caveolin- and lipid raft-dependent endocytosis, which allowed the transfer of miR-222-3p. Exosomic miR-222-3p enhanced the proliferation, gemcitabine resistance, migration, invasion, and anti-anoikis of parental sensitive cells by directly targeting the promoter of SOCS3. In addition, a higher level of exosomic miR-222-3p in sera usually predicted worse prognosis in NSCLC patients. CONCLUSION: Our data demonstrate that exosomic-miR-222-3p functions as a principal regulator of gemcitabine resistance and malignant characteristics by targeting SOCS3. The exosomic miR-222-3p level in sera may be a potential prognostic biomarker for predicting gemcitabine sensitivity in NSCLC patients.

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Fig. 1 Gemcitabine-resistant cells shed exosomes that readily enter A549-P cells. a A549-P/GR cells were treated with increasing concentrations of gemcitabine for 72 h. Cell growth was analyzed by MTS assay. Data represent at least three experiments performed in triplicate. Error bars represent ± SD. *P < 0.01; **P < 0.001. b A549-P/GR cells were treated with gemcitabine (1 μM) and examined by colony formation assay. c The presence of TSG101, CD81, and Alix in 15 μg of exosomes was analyzed by western blot. d Exosomes were observed by transmission electron microscopy. Scale bar, 100 μm. e Quantification of exosomes expelled from equal numbers of A549-P/GR cells by BCA analysis. f A549-P cells were treated with different pharmacological inhibitors of endocytic pathways for 2 h. A549-GR–derived exosomes (GR-Exo) were dyed with PKH67 (green) and incubated with pretreated cells for 4 h, and exosome uptake was viewed under confocal microscopy
Fig. 1 Gemcitabine-resistant cells shed exosomes that readily enter A549-P cells. a A549-P/GR cells were treated with increasing concentrations of gemcitabine for 72 h. Cell growth was analyzed by MTS assay. Data represent at least three experiments performed in triplicate. Error bars represent ± SD. *P < 0.01; **P < 0.001. b A549-P/GR cells were treated with gemcitabine (1 μM) and examined by colony formation assay. c The presence of TSG101, CD81, and Alix in 15 μg of exosomes was analyzed by western blot. d Exosomes were observed by transmission electron microscopy. Scale bar, 100 μm. e Quantification of exosomes expelled from equal numbers of A549-P/GR cells by BCA analysis. f A549-P cells were treated with different pharmacological inhibitors of endocytic pathways for 2 h. A549-GR–derived exosomes (GR-Exo) were dyed with PKH67 (green) and incubated with pretreated cells for 4 h, and exosome uptake was viewed under confocal microscopyCC BY · PubMed Central
Fig. 2 GR-Exo deliver miRNA-222-3p into recipient cells, enhance gemcitabine resistance, promote cell metastasis and proliferation. a Heatmap of differential miRNA expression between A549-GR and A549-P exosomes. Gene expression data were obtained using a human microRNA array. Expression values shown are mean centered. Red: increased expression, Green: decreased expression. b miR-222-3p expression was detected in A549-P/GR cells and A549-P/GR–derived exosomes by qRT-PCR. c miR-222-3p expression was detected in A549-P cells after co-incubation with P-Exo or GR-Exo for 24 h by qRT-PCR. d miR-222-3p expression was detected in H460 and H157 cells after co-incubation with P-Exo or GR-Exo by qRT-PCR. e The growth rates of A549-P and A549-P-KD cells in the absence or presence of GR-Exo were assessed using MTS assay. f Cell growth was assessed by MTS after treatment with gemcitabine for 72 h in the absence or presence of P-Exo or GR-Exo. g The migration of exosome-treated cells was assessed using wound-healing assay. Right, quantitative analysis of invasive cells. h Transwell assay was performed to assess cell invasion. Right, quantitative analysis of scratch wound closure. i Soft agar assay was performed to examine the anchorage-independent survival of cells. Right, quantitative analysis of cell clones. Data represent at least three experiments performed in triplicate. *P < 0.05; **P < 0.01
Fig. 2 GR-Exo deliver miRNA-222-3p into recipient cells, enhance gemcitabine resistance, promote cell metastasis and proliferation. a Heatmap of differential miRNA expression between A549-GR and A549-P exosomes. Gene expression data were obtained using a human microRNA array. Expression values shown are mean centered. Red: increased expression, Green: decreased expression. b miR-222-3p expression was detected in A549-P/GR cells and A549-P/GR–derived exosomes by qRT-PCR. c miR-222-3p expression was detected in A549-P cells after co-incubation with P-Exo or GR-Exo for 24 h by qRT-PCR. d miR-222-3p expression was detected in H460 and H157 cells after co-incubation with P-Exo or GR-Exo by qRT-PCR. e The growth rates of A549-P and A549-P-KD cells in the absence or presence of GR-Exo were assessed using MTS assay. f Cell growth was assessed by MTS after treatment with gemcitabine for 72 h in the absence or presence of P-Exo or GR-Exo. g The migration of exosome-treated cells was assessed using wound-healing assay. Right, quantitative analysis of invasive cells. h Transwell assay was performed to assess cell invasion. Right, quantitative analysis of scratch wound closure. i Soft agar assay was performed to examine the anchorage-independent survival of cells. Right, quantitative analysis of cell clones. Data represent at least three experiments performed in triplicate. *P < 0.05; **P < 0.01CC BY · PubMed Central
Fig. 3 SOCS3 is the direct target of miR-222-3p. a SOCS3 expression was examined by western blotting. b Sequence alignment of miR-222-3p with reverse complementary miR-222-3p (rcmiR-222-3p), SOCS3 (SOCS3 3’UTR), mutant rcmiR-222-3p (mrcmiR-222-3p), and mutant SOCS3 (mSOCS3 3’UTR). c SOCS3 expression in A549-P cells after transfection with miR-222-3p mimic or inhibitor was examined by western blotting. d The promoter activities of SOCS3 were measured using the dual-Luciferase reporter assay after transfection of A549-P cells alone or in the presence of miR-222-3p mimic or inhibitor. Data are representative of three experiments. Error bars represent ±SD. *P < 0.05; **P < 0.01 vs. vector alone group. e Expression levels of SOCS3/JAK2/Stat3 and Bcl-2/Bax after transfection with miR-222-3p mimic and inhibitor were assessed by western blotting
Fig. 3 SOCS3 is the direct target of miR-222-3p. a SOCS3 expression was examined by western blotting. b Sequence alignment of miR-222-3p with reverse complementary miR-222-3p (rcmiR-222-3p), SOCS3 (SOCS3 3’UTR), mutant rcmiR-222-3p (mrcmiR-222-3p), and mutant SOCS3 (mSOCS3 3’UTR). c SOCS3 expression in A549-P cells after transfection with miR-222-3p mimic or inhibitor was examined by western blotting. d The promoter activities of SOCS3 were measured using the dual-Luciferase reporter assay after transfection of A549-P cells alone or in the presence of miR-222-3p mimic or inhibitor. Data are representative of three experiments. Error bars represent ±SD. *P < 0.05; **P < 0.01 vs. vector alone group. e Expression levels of SOCS3/JAK2/Stat3 and Bcl-2/Bax after transfection with miR-222-3p mimic and inhibitor were assessed by western blottingCC BY · PubMed Central
Fig. 4 Exogenous overexpression of SOCS3 rescues A549-P cells from GR-Exo–induced stronger malignancy. A549-P or A549-GR cells were transfected with GV-144-SOCS3 plasmid and treated with GR-Exo. a SOCS3 expression was assessed by western blotting. b Cell viability was assessed by MTS after co-treatment with gemcitabine for 72 h. Data are representative of three experiments. Error bars represent ±SD. *P < 0.05; **P < 0.01 vs. control. c Wound-healing assay and d Transwell assay were performed to assess cell migration and invasion. Lower, quantitative analysis of scratch wound closure (c) or cell invasion (d). Data represented at least three experiments performed in triplicate. *P < 0.05; **P < 0.01
Fig. 4 Exogenous overexpression of SOCS3 rescues A549-P cells from GR-Exo–induced stronger malignancy. A549-P or A549-GR cells were transfected with GV-144-SOCS3 plasmid and treated with GR-Exo. a SOCS3 expression was assessed by western blotting. b Cell viability was assessed by MTS after co-treatment with gemcitabine for 72 h. Data are representative of three experiments. Error bars represent ±SD. *P < 0.05; **P < 0.01 vs. control. c Wound-healing assay and d Transwell assay were performed to assess cell migration and invasion. Lower, quantitative analysis of scratch wound closure (c) or cell invasion (d). Data represented at least three experiments performed in triplicate. *P < 0.05; **P < 0.01CC BY · PubMed Central
Fig. 5 Knockdown of miR-222-3p in A549-P/GR cells suppresses tumor growth and lung metastasis in vivo. Lung metastasis models in SCID mice were generated by using luciferase-labeled A549-P/GR cells with lentivirus miR-222-3p KD or normal control (NC) and treated with PBS (control) or GR-Exo (n = 8). a miR-222-3p expression was assessed by qRT-PCR. b SOCS3 expression was assessed by western blotting. Two samples were selected from each group randomly (n = 8) (labeled as 1,2), and all samples were sufficiently mixed as a pool (labeled as mix). c The incidence of metastasis in the lung or other organs in each group. d Luminescence was assessed in 8 mice of each group at 6 weeks after injection. e To quantify the metastatic mass, the photon counts per second were recorded (***P < 0.001). f The lungs of mice from each group were removed, and lung metastasis was evaluated by hematoxylin and eosin (H&E) staining
Fig. 5 Knockdown of miR-222-3p in A549-P/GR cells suppresses tumor growth and lung metastasis in vivo. Lung metastasis models in SCID mice were generated by using luciferase-labeled A549-P/GR cells with lentivirus miR-222-3p KD or normal control (NC) and treated with PBS (control) or GR-Exo (n = 8). a miR-222-3p expression was assessed by qRT-PCR. b SOCS3 expression was assessed by western blotting. Two samples were selected from each group randomly (n = 8) (labeled as 1,2), and all samples were sufficiently mixed as a pool (labeled as mix). c The incidence of metastasis in the lung or other organs in each group. d Luminescence was assessed in 8 mice of each group at 6 weeks after injection. e To quantify the metastatic mass, the photon counts per second were recorded (***P < 0.001). f The lungs of mice from each group were removed, and lung metastasis was evaluated by hematoxylin and eosin (H&E) stainingCC BY · PubMed Central

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Authors
Wei, F., Ma, C., Zhou, T., Dong, X., Luo, Q., Geng, L., Ding, L., Zhang, Y., Zhang, L., Li, N., Li, Y., & Liu, Y.
Affiliations
  • First Hospital of Jilin University (CN)
  • Academy of Military Medical Sciences (CN)
  • Jilin University (CN)
Corresponding author
Liu, Yan
ORCID
Venue
Molecular Cancer
Publisher
Springer Science and Business Media LLC
Published
2017-07-25
Volume
16
Issue
1
Pages
132-132
ISSN
1476-4598 (ISSN-L)
DOI
10.1186/s12943-017-0694-8
PMC
PMC5526308
OpenAlex
W2736949652
Cited by
245
References
52
Citations by year
2026: 11 · 2025: 19 · 2024: 26 · 2023: 25 · 2022: 32 · 2021: 45 · 2020: 43 · 2019: 23 · 2018: 17 · 2017: 4
FWCI
9.72
Type
journal article
Language
en
Update notices
correction (2021-02-17)
Open access
Licence
Locations
  • Molecular Cancer (publishedVersion, cc-by)
  • 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 (81301884, 81401883, 81302173)
  • Natural Science Foundation of Jilin Province (20140520036JH, 2016041052GH, 20140414027GH, 20160101053JC)
  • Jilin University (450060521279)
  • China Postdoctoral Science Foundation (2016M590264)
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
  • Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
Keywords
exosomes, gemcitabine resistance, non-small cell lung cancer, miR-222-3p, SOCS3, caveolin-dependent endocytosis, lipid rafts, metastasis, anoikis resistance, cell proliferation and invasion, serum biomarkers, prognostic factors, chemotherapy response
MeSH terms
Gemcitabine, Animals, Carcinoma, Non-Small-Cell Lung, Cell Movement, Deoxycytidine, Female, Humans, Lung Neoplasms, Male, Middle Aged, Neoplasm Recurrence, Local, Gene Expression Regulation, Neoplastic, Mice, SCID, 3' Untranslated Regions, MicroRNAs, Cell Line, Tumor, Cell Proliferation, Mice, Exosomes

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