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Cited by 222Extracellular vesicles in diseaseMicroRNA in disease regulationCancer Cells and Metastasis

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Exosomal miR-21-5p derived from gastric cancer promotes peritoneal metastasis via mesothelial-to-mesenchymal transition

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Li, Qiang, Li, Bowen, Li, Qing, Wei, Song, He, Zhongyuan, Huang, Xiaoxu, Wang, Lu, Xia, Yiwen, Xu, Zhipeng, Li, Zheng, Wang, Weizhi, Yang, Li, Zhang, Diancai, Xu, Zekuan·2018·Cell Death & Disease
DOI 10.1038/s41419-018-0928-8PMC PMC6113299
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Abstract

Peritoneal metastasis is a primary metastatic route for gastric cancers, and the mechanisms underlying this process are still unclear. Peritoneal mesothelial cells (PMCs) undergo mesothelial-to-mesenchymal transition (MMT) to provide a favorable environment for metastatic cancer cells. In this study, we investigated how the exosomal miR-21-5p induces MMT and promotes peritoneal metastasis. Gastric cancer (GC)-derived exosomes were identified by transmission electron microscopy and western blot analysis, then the uptake of exosomes was confirmed by PKH-67 staining. The expression of miR-21-5p and SMAD7 were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, and the interactions between miR-21-5p and its target genes SMAD7 were confirmed by Luciferase reporter assays. The MMT of PMCs was determined by invasion assays, adhesion assays, immunofluorescent assay, and western blot. Meanwhile, mouse model of tumor peritoneal dissemination model was performed to investigate the role of exosomal miR-21-5p in peritoneal metastasis in vivo. We found that PMCs could internalize GC-derived exosomal miR-21-5p and led to increased levels of miR-21-5p in PMCs. Through various types of in vitro and in vivo assays, we confirmed that exosomal miR-21-5p was able to induce MMT of PMCs and promote tumor peritoneal metastasis. Moreover, our study revealed that this process was promoted by exosomal miR-21-5p through activating TGF-β/Smad pathway via targeting SMAD7. Altogether, our data suggest that exosomal miR-21-5p induces MMT of PMCs and promote cancer peritoneal dissemination by targeting SMAD7. The exosomal miR-21-5p may be a novel therapeutic target for GC peritoneal metastasis.

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Figures

Fig. 1 Characterization of GC cells-derived exosomes and internalization. a Exosomes purified from culture supernatant of the four GC cells and GES-1 cells were detected by TEM (Scale bar, 200 nm). b Exosomes marker proteins CD63 and TSG101 were identified by western blot. Calnexin was used as an internal reference. c Exosomes purified from culture supernatant of the four GC cells and GES-1 cells were labeled by PKH67, and HMrSV5 co-cultured with these exosomes or non-exosomes were observed under confocal microscopy (Scale bar = 20 µm). Merge-2 group was a number of pictures captured at lower magnification than merge-1. Non-exosomes group was used as the negative control. Each experiment was repeated at least three times
Fig. 1 Characterization of GC cells-derived exosomes and internalization. a Exosomes purified from culture supernatant of the four GC cells and GES-1 cells were detected by TEM (Scale bar, 200 nm). b Exosomes marker proteins CD63 and TSG101 were identified by western blot. Calnexin was used as an internal reference. c Exosomes purified from culture supernatant of the four GC cells and GES-1 cells were labeled by PKH67, and HMrSV5 co-cultured with these exosomes or non-exosomes were observed under confocal microscopy (Scale bar = 20 µm). Merge-2 group was a number of pictures captured at lower magnification than merge-1. Non-exosomes group was used as the negative control. Each experiment was repeated at least three timesCC BY · PubMed Central
Fig. 2 GC-derived exosomes induce MMT of HMrSV5 cells in vivo and promote peritoneal metastasis. a Invasion assays of HMrSV5 cells which were pretreated with 200 µg exosomes isolated from culture supernatant of four GC cells and GES-1 cells. Scale bar = 100 µm. b Adhesion assays of HMrSV5 cells which were pretreated with 200 µg exosomes. Scale bar = 100 µm. Calcein AM labeled BGC823 cells were added to investigate the adhesion ablitity of HMrSV5. c Bioluminescence images of tumor peritoneal dissemination. d MMT protein markers (Vimentin and α-SMA) were identified by immunofluorescence assays in exosomes-treated group and control group. Scale bar = 50 µm. e Western blot was performed to further confirm the MMT protein markers (including E-cadherin, α-SMA, and Vimentin). GES-1 cells-derived exosomes were used as the negative control in vivo and in vitro. GAPDH was used as an internal reference for western blot. Each experiment was repeated at least three times. All the data were expressed as mean ± SEM, and the results of multiple comparisons were corrected with Bonferroni method (Student’s t-test *P < 0.05, **P < 0.01, ***P < 0.001)
Fig. 2 GC-derived exosomes induce MMT of HMrSV5 cells in vivo and promote peritoneal metastasis. a Invasion assays of HMrSV5 cells which were pretreated with 200 µg exosomes isolated from culture supernatant of four GC cells and GES-1 cells. Scale bar = 100 µm. b Adhesion assays of HMrSV5 cells which were pretreated with 200 µg exosomes. Scale bar = 100 µm. Calcein AM labeled BGC823 cells were added to investigate the adhesion ablitity of HMrSV5. c Bioluminescence images of tumor peritoneal dissemination. d MMT protein markers (Vimentin and α-SMA) were identified by immunofluorescence assays in exosomes-treated group and control group. Scale bar = 50 µm. e Western blot was performed to further confirm the MMT protein markers (including E-cadherin, α-SMA, and Vimentin). GES-1 cells-derived exosomes were used as the negative control in vivo and in vitro. GAPDH was used as an internal reference for western blot. Each experiment was repeated at least three times. All the data were expressed as mean ± SEM, and the results of multiple comparisons were corrected with Bonferroni method (Student’s t-test *P < 0.05, **P < 0.01, ***P < 0.001)CC BY · PubMed Central
Fig. 3 GC-derived exosomes transfer miR-21-5p from donor cells to recipient cells. a The expression levels of miR-21-5p in TCGA dataset between GC tissues and normal tissues. All normalized expression values were expressed in log2((RPM + 1)). b The expression levels of miR-21-5p in 49 paired GC tissues and adjacent normal tissues. All normalized expression values were expressed in log22 −ΔΔCT. c qRT-PCR detection of miR-21-5p expression in exosomes derived from four GC cell lines and GES-1 cells. GES-1 cells-derived exosomes were used as the negative control. d qRT-PCR detection of miR-21-5p expression in HMrSV5 co-cultured with MGC803-derived exosomes and GES-1 cells-derived exosomes (negative control). e Exosomes with Cy3-labeled miR-21-5p or without Cy3-labeled miR-21-5p were added to HMrSV5 cells. MiR-21-5p without Cy3-label was used as the negative control. The fluorescence signals were detected under confocal microscope. Scale bar = 20 µm. f The levels of miR-21-5p in HMrSV5 cells transfected with miR-21-5p-NC or miR-21-inhibitor and in the related exosomes. g qRT-PCR detection of miR-21-5p expression in HMrSV5 cells co-cultured with exosomes derived from GES-1 cells or MGC803 cells transfected with miR-21-5p-NC or miR-21-5p-inhibitor. h Immunofluorescence assays were performed to measure the MMT protein markers (Vimentin and α-SMA). Scale bar = 50 µm. MiR-21-5p-NC group was used as the negative control. Each experiment was repeated at least three times. All the data were expressed as mean ± SEM, and the results of multiple comparisons were corrected with Bonferroni method (Student’s t-test *P < 0.05, **P < 0.01, ***P < 0.001)
Fig. 3 GC-derived exosomes transfer miR-21-5p from donor cells to recipient cells. a The expression levels of miR-21-5p in TCGA dataset between GC tissues and normal tissues. All normalized expression values were expressed in log2((RPM + 1)). b The expression levels of miR-21-5p in 49 paired GC tissues and adjacent normal tissues. All normalized expression values were expressed in log22 −ΔΔCT. c qRT-PCR detection of miR-21-5p expression in exosomes derived from four GC cell lines and GES-1 cells. GES-1 cells-derived exosomes were used as the negative control. d qRT-PCR detection of miR-21-5p expression in HMrSV5 co-cultured with MGC803-derived exosomes and GES-1 cells-derived exosomes (negative control). e Exosomes with Cy3-labeled miR-21-5p or without Cy3-labeled miR-21-5p were added to HMrSV5 cells. MiR-21-5p without Cy3-label was used as the negative control. The fluorescence signals were detected under confocal microscope. Scale bar = 20 µm. f The levels of miR-21-5p in HMrSV5 cells transfected with miR-21-5p-NC or miR-21-inhibitor and in the related exosomes. g qRT-PCR detection of miR-21-5p expression in HMrSV5 cells co-cultured with exosomes derived from GES-1 cells or MGC803 cells transfected with miR-21-5p-NC or miR-21-5p-inhibitor. h Immunofluorescence assays were performed to measure the MMT protein markers (Vimentin and α-SMA). Scale bar = 50 µm. MiR-21-5p-NC group was used as the negative control. Each experiment was repeated at least three times. All the data were expressed as mean ± SEM, and the results of multiple comparisons were corrected with Bonferroni method (Student’s t-test *P < 0.05, **P < 0.01, ***P < 0.001)CC BY · PubMed Central
Fig. 4 MiR-21-5p induces MMT in HMrSV5 cells in vitro. a qRT-PCR detection of miR-21-5p expression in HMrSV5 transfected with miR-21-5p-mimics, mimics-NC, miR-21-5p-inhibitor and inhibitor-NC. Mimics-NC was the negative control for miR-21-5p-mimics and inhibitor-NC was the negative control for miR-21-5p-inhibitor. b, c Adhesion (Scale bar = 100 µm) and invasion (Scale bar = 100 µm) assays showed the effect of miR-21-5p on the HMrSV5 in vitro. d, e Western blot assays were performed to confirm the MMT of HMrSV5. GAPDH was used as an internal control. f Immunofluorescence assays were performed to confirm the MMT of HMrSV5. Scale bar = 50 µm. Each experiment was repeated at least three times. All the data were expressed as the mean ± SEM (Student’s t-test *P < 0.05, **P < 0.01, ***P < 0.001)
Fig. 4 MiR-21-5p induces MMT in HMrSV5 cells in vitro. a qRT-PCR detection of miR-21-5p expression in HMrSV5 transfected with miR-21-5p-mimics, mimics-NC, miR-21-5p-inhibitor and inhibitor-NC. Mimics-NC was the negative control for miR-21-5p-mimics and inhibitor-NC was the negative control for miR-21-5p-inhibitor. b, c Adhesion (Scale bar = 100 µm) and invasion (Scale bar = 100 µm) assays showed the effect of miR-21-5p on the HMrSV5 in vitro. d, e Western blot assays were performed to confirm the MMT of HMrSV5. GAPDH was used as an internal control. f Immunofluorescence assays were performed to confirm the MMT of HMrSV5. Scale bar = 50 µm. Each experiment was repeated at least three times. All the data were expressed as the mean ± SEM (Student’s t-test *P < 0.05, **P < 0.01, ***P < 0.001)CC BY · PubMed Central
Fig. 5 MiR-21-5p directly targets SMAD7-3′-UTR in HMrSV5 cells. a Luciferase reporter assay results indicated that miR-21-5p directly bind to SMAD7 3’UTR. b Western blot was performed to measure the protein levels of SMAD7 in different groups. c qRT-PCR detection of SMAD7 mRNA in HMrSV5 cells transfected with miR-21-5p-mimics, mimics-NC, miR-21-5p-inhibitor and inhibitor-NC. d Western blot analysis for the protein levels of SMAD2/3, p-SMAD2/3, and TβRI in different groups. GAPDH was used as an internal reference. Each experiment was repeated at least three times. All the data were expressed as mean ± SEM (Student’s t-test *P < 0.05, **P < 0.01, ***P < 0.001)
Fig. 5 MiR-21-5p directly targets SMAD7-3′-UTR in HMrSV5 cells. a Luciferase reporter assay results indicated that miR-21-5p directly bind to SMAD7 3’UTR. b Western blot was performed to measure the protein levels of SMAD7 in different groups. c qRT-PCR detection of SMAD7 mRNA in HMrSV5 cells transfected with miR-21-5p-mimics, mimics-NC, miR-21-5p-inhibitor and inhibitor-NC. d Western blot analysis for the protein levels of SMAD2/3, p-SMAD2/3, and TβRI in different groups. GAPDH was used as an internal reference. Each experiment was repeated at least three times. All the data were expressed as mean ± SEM (Student’s t-test *P < 0.05, **P < 0.01, ***P < 0.001)CC BY · PubMed Central
Fig. 6 MiR-21-5p promotes MMT of HMrSV5 via targeting SMAD7. a, b To investigate the rescue function of SMAD7, adhesion (Scale bar = 100 µm) and invasion (Scale bar = 100 µm) assays were performed in HMrSV5-miR-21-5p-mimics or HMrSV5-miR-21-5p-inhibitor cells which were transfected with LV-SMAD7 or sh-SMAD7 without its 3’UTR. The negative controls for miR-21-5p-mimics + LV-SMAD7 and miR-21-5p-inhibitor + sh-SMAD7 were miR-21-5p-mimics and miR-21-5p-inhibitor, respectively. c, d The alteration of protein levels (including SMAD7, E-cadherin, α-SMA and Vimentin) were meatured by western blot. GAPDH was used as a loading control. Each experiment was repeated at least three times. All the data were expressed as the mean ± SEM, and the results of multiple comparisons were corrected with Bonferroni method (Student’s t-test *P < 0.05, **P < 0.01, ***P < 0.001)
Fig. 6 MiR-21-5p promotes MMT of HMrSV5 via targeting SMAD7. a, b To investigate the rescue function of SMAD7, adhesion (Scale bar = 100 µm) and invasion (Scale bar = 100 µm) assays were performed in HMrSV5-miR-21-5p-mimics or HMrSV5-miR-21-5p-inhibitor cells which were transfected with LV-SMAD7 or sh-SMAD7 without its 3’UTR. The negative controls for miR-21-5p-mimics + LV-SMAD7 and miR-21-5p-inhibitor + sh-SMAD7 were miR-21-5p-mimics and miR-21-5p-inhibitor, respectively. c, d The alteration of protein levels (including SMAD7, E-cadherin, α-SMA and Vimentin) were meatured by western blot. GAPDH was used as a loading control. Each experiment was repeated at least three times. All the data were expressed as the mean ± SEM, and the results of multiple comparisons were corrected with Bonferroni method (Student’s t-test *P < 0.05, **P < 0.01, ***P < 0.001)CC BY · PubMed Central
Fig. 7 MiR-21-5p promotes GC peritoneal metastasis by inducing MMT in vivo. a Western blot was performed to analyse the expression levels of SMAD7, E-cadherin, α-SMA, and Vimentin in peritoneal tissues from different groups. GAPDH was used as a loading control. b, d Bioluminescence images of tumor peritoneal dissemination in different groups, and the luciferase activity was analyzed. c, e Representative images were captured from different groups, and tumors were outlined in white. f HE stain for tumor tissues from each groups (Scale bar = 50 µm, 100X magnification; Scale bar = 100 µm, 400X magnification). Each experiment was repeated at least three times. All the data were expressed as mean ± SEM (Student’s t-test *P < 0.05, **P < 0.01, ***P < 0.001)
Fig. 7 MiR-21-5p promotes GC peritoneal metastasis by inducing MMT in vivo. a Western blot was performed to analyse the expression levels of SMAD7, E-cadherin, α-SMA, and Vimentin in peritoneal tissues from different groups. GAPDH was used as a loading control. b, d Bioluminescence images of tumor peritoneal dissemination in different groups, and the luciferase activity was analyzed. c, e Representative images were captured from different groups, and tumors were outlined in white. f HE stain for tumor tissues from each groups (Scale bar = 50 µm, 100X magnification; Scale bar = 100 µm, 400X magnification). Each experiment was repeated at least three times. All the data were expressed as mean ± SEM (Student’s t-test *P < 0.05, **P < 0.01, ***P < 0.001)CC BY · PubMed Central

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Metadata

Authors
Li, Q., Li, B., Li, Q., Wei, S., He, Z., Huang, X., Wang, L., Xia, Y., Xu, Z., Li, Z., Wang, W., Yang, L., Zhang, D., & Xu, Z.
Affiliations
Jiangsu Province Hospital (CN)
Corresponding author
Zhang, Diancai
ORCID
Venue
Cell Death & Disease
Publisher
Springer Science and Business Media LLC
Published
2018-08-28
Volume
9
Issue
9
Pages
854-854
ISSN
2041-4889 (ISSN-L)
DOI
10.1038/s41419-018-0928-8
PMC
PMC6113299
OpenAlex
W2889190369
Cited by
219
References
47
Citations by year
2026: 11 · 2025: 21 · 2024: 31 · 2023: 34 · 2022: 45 · 2021: 46 · 2020: 17 · 2019: 14
FWCI
7.03
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Death and Disease (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)
Funders
  • National Natural Science Foundation of China (JX10231801, 30901443)
  • Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, JX10231801, JX 10231801)
  • National Institutes of Health
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
gastric cancer, peritoneal carcinomatosis, exosomes, miR-21-5p, mesothelial-to-mesenchymal transition, peritoneal mesothelial cells, Smad7, TGF-β/Smad signaling, extracellular vesicles, tumor microenvironment
MeSH terms
Animals, Cell Movement, Epithelium, Female, Humans, Male, Mice, Nude, Middle Aged, Neoplasm Metastasis, Peritoneal Neoplasms, Peritoneum, Stomach Neoplasms, Signal Transduction, Gene Expression Regulation, Neoplastic, Transforming Growth Factor beta, MicroRNAs, Cell Line, Tumor, Cell Proliferation, Mice, Smad7 Protein, Exosomes, Epithelial-Mesenchymal Transition

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