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Lung cancer-derived exosomal miR-132-3p contributed to interstitial lung disease development

Fang, Sufang, Wang, Ting, Weng, Ling, Han, Ximei, Zheng, Rongshan, Zhang, Hongying·2023·World Journal of Surgical Oncology
DOI 10.1186/s12957-023-03095-6PMC PMC10349414
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Abstract

Purpose: Interstitial lung diseases (ILDs) have high morbidity and mortality and poor prognosis. The significance of microRNAs (miRNAs) was highlighted in ILDs development. Currently, we attempted to confirm the functions of lung cancer-derived exosomal miR-132-3p and reveal the underlying mechanism. Method: Characteristics of exosomes were verified by transmission electron microscope (TEM), nanoparticle tracking analysis, and Western blot assay. Exosome uptake for the normal human lung fibroblasts (NHLF) was assessed using a PKH67 staining assay. MTT and colony formation assays were applied to examine the proliferation abilities of NHLF. The interaction between miR-132-3p and sprouty1 (SPRY1) was confirmed by a luciferase reporter assay. Results: Lung cancer-derived exosomes promoted normal human lung fibroblast activation. Exosome inhibitor GW4869 reversed the effects of Exo on NHLF. Subsequently, miR-132-3p in lung cancer-derived exosomes activated the normal human lung fibroblast and promoted interstitial lung disease development ex vivo. Next, SPRY1 was verified to be the binding protein of miR-132-3p, and sh-SPRY1 abrogated the effects of the miR-132-3p inhibitor on NHLF. Conclusion: Exosomal miR-132-3p from A549 cells accelerated the development of interstitial lung disease through binding to SPRY1, which might serve as an important target for ILDs.

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Figures

Fig. 1 The exosomes from A549 cells were identified. A The structure of exosomes from A549 cells was identified by TEM. B The diameter for the exosomes from A549 cells was tracked by NTA. C The representative exosomal markers such as Alix, TSG101, CD9, CD63, calnexin and CD63, and tubulin protein expressions were detected in lung cancer-derived exosomes and corresponding cells
Fig. 1 The exosomes from A549 cells were identified. A The structure of exosomes from A549 cells was identified by TEM. B The diameter for the exosomes from A549 cells was tracked by NTA. C The representative exosomal markers such as Alix, TSG101, CD9, CD63, calnexin and CD63, and tubulin protein expressions were detected in lung cancer-derived exosomes and corresponding cellsCC BY · PubMed Central
Fig. 2 The exosomes from A549 cells activated the normal human lung fibroblast. A The green fluorescence levels were detected for NHLF co-cultured with PKH67-labeled exosomes from A549 cells. B MTT assay tested the viability of NHLF treated with exosomes or exosomes + GW4869. C The proliferation abilities of NHLF, which were incubated with exosomes or exosomes + GW4869, were checked by clone formation assay. D The expression levels of proliferation-related proteins (Ki-67 and PCNA) were tested for control, exo and exo + GW4869 group. E Flow cytometry assay was performed to analyze the apoptosis rates in all groups. F Western blot analysis of the pro-/anti-apoptosis proteins (Bax and Caspase-3 and Bcl-2) was performed. G–H α-SMA, FN, TGF-β, COL1A1, COL3A1, CXCL12, IL-1β, IL-6, and IL-8 mRNA expressions were tested by qRT-PCR analysis. Data were presented as mean ± SD of three independent experiments. ***P < 0.001; **P < 0.01
Fig. 2 The exosomes from A549 cells activated the normal human lung fibroblast. A The green fluorescence levels were detected for NHLF co-cultured with PKH67-labeled exosomes from A549 cells. B MTT assay tested the viability of NHLF treated with exosomes or exosomes + GW4869. C The proliferation abilities of NHLF, which were incubated with exosomes or exosomes + GW4869, were checked by clone formation assay. D The expression levels of proliferation-related proteins (Ki-67 and PCNA) were tested for control, exo and exo + GW4869 group. E Flow cytometry assay was performed to analyze the apoptosis rates in all groups. F Western blot analysis of the pro-/anti-apoptosis proteins (Bax and Caspase-3 and Bcl-2) was performed. G–H α-SMA, FN, TGF-β, COL1A1, COL3A1, CXCL12, IL-1β, IL-6, and IL-8 mRNA expressions were tested by qRT-PCR analysis. Data were presented as mean ± SD of three independent experiments. ***P < 0.001; **P < 0.01CC BY · PubMed Central
Fig. 3 MiR-132-3p in exosomes activated the normal human lung fibroblast. A MiR-132-3p had a high expression in the non-small-cell lung tumor-derived exosomal miRNAs. B The qRT-PCR tested the expression of miR-132-3p in exosomes from A549 cells and the corresponding cells. C In NHLF, the transfection efficiency of miR-132-3p mimic or inhibitor was evaluated. D MTT assays tested the cell viability in miR-132-3p mimic, miR-132-3p inhibitor, and miR-NC groups. E Clone formation assays tested the cell proliferation in all groups. F Flow cytometry analyzed the apoptosis rates in all groups. G Western blot assay was performed to analyze the expression levels of Bax, Bcl-2, and cleaved caspase-3 in NHLF transfected with miR-132-3p mimic, miR-132-3p inhibitor, or miR-NC. H-I. The mRNA expressions, including α-SMA, FN, TGF-β, COL1A1, COL3A1, CXCL12, and IL-1β/6/8, were tested by qRT-PCR analysis. Data were presented as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01, ***P < 0.001
Fig. 3 MiR-132-3p in exosomes activated the normal human lung fibroblast. A MiR-132-3p had a high expression in the non-small-cell lung tumor-derived exosomal miRNAs. B The qRT-PCR tested the expression of miR-132-3p in exosomes from A549 cells and the corresponding cells. C In NHLF, the transfection efficiency of miR-132-3p mimic or inhibitor was evaluated. D MTT assays tested the cell viability in miR-132-3p mimic, miR-132-3p inhibitor, and miR-NC groups. E Clone formation assays tested the cell proliferation in all groups. F Flow cytometry analyzed the apoptosis rates in all groups. G Western blot assay was performed to analyze the expression levels of Bax, Bcl-2, and cleaved caspase-3 in NHLF transfected with miR-132-3p mimic, miR-132-3p inhibitor, or miR-NC. H-I. The mRNA expressions, including α-SMA, FN, TGF-β, COL1A1, COL3A1, CXCL12, and IL-1β/6/8, were tested by qRT-PCR analysis. Data were presented as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01, ***P < 0.001CC BY · PubMed Central
Fig. 4 MiR-132-3p in exosomes aggravated interstitial lung disease development in vivo. A Representative histological lung sections were subjected to H&E and Masson staining and immunohistochemical analysis for α-SMA from saline, BLM, BLM + Exo, BLM + miR-NC-exo, and BLM + miR-132-3p inhibitor-exo groups. B In all groups, α-SMA, COL1A1, COL3A1, and TGF-β1 mRNA expressions were tested by qRT-PCR analysis. C In saline, BLM, BLM + Exo, BLM + miR-NC-exo, and BLM + miR-132-3p inhibitor-exo groups, the mRNA expressions of IL-1β/6/8 were tested. Data were presented as mean ± SD of three independent experiments. ***P < 0.001; **P < 0.01
Fig. 4 MiR-132-3p in exosomes aggravated interstitial lung disease development in vivo. A Representative histological lung sections were subjected to H&E and Masson staining and immunohistochemical analysis for α-SMA from saline, BLM, BLM + Exo, BLM + miR-NC-exo, and BLM + miR-132-3p inhibitor-exo groups. B In all groups, α-SMA, COL1A1, COL3A1, and TGF-β1 mRNA expressions were tested by qRT-PCR analysis. C In saline, BLM, BLM + Exo, BLM + miR-NC-exo, and BLM + miR-132-3p inhibitor-exo groups, the mRNA expressions of IL-1β/6/8 were tested. Data were presented as mean ± SD of three independent experiments. ***P < 0.001; **P < 0.01CC BY · PubMed Central
Fig. 5 miR-132-3p interacted with SPRY1. A Bioinformatic analysis of the predicted interaction sites between miR-132-3p and SPRY1 was performed by TargetScan analysis. B Luciferase reporter assay was used to assess the relationship between miR-132-3p and SPRY1. C The mRNA and protein levels of SPRY1 were assessed. D In the isolated exosomes and A549 cells, the mRNA of SPRY1 was assessed. Data were presented as mean ± SD of three independent experiments. **P < 0.01
Fig. 5 miR-132-3p interacted with SPRY1. A Bioinformatic analysis of the predicted interaction sites between miR-132-3p and SPRY1 was performed by TargetScan analysis. B Luciferase reporter assay was used to assess the relationship between miR-132-3p and SPRY1. C The mRNA and protein levels of SPRY1 were assessed. D In the isolated exosomes and A549 cells, the mRNA of SPRY1 was assessed. Data were presented as mean ± SD of three independent experiments. **P < 0.01CC BY · PubMed Central
Fig. 6 Inhibition of miR-132-3p abrogated the progression of interstitial lung disease via targeting SPRY1 in NHLF. A The knockdown efficiency of SPRY1 in NHLF was tested by qRT-PCR and Western blot assays. B Cell viabilities were assessed in NHLF transfected with miR-NC, miR-132-3p inhibitor, miR-132-3p inhibitor + sh-NC, and miR-132-3p inhibitor + sh-SPRY1. C Cell colony numbers were detected in all groups. D Flow cytometry analyzed the apoptosis rate in all groups. E The anti-/pro-apoptosis protein expressions were assessed by Western blot assay. F-G The fibrosis/collagen-associated genes (α-SMA, TGF-β1, COL1A1, and COL3A1), and many pro-inflammatory mediators (IL-1β/6/8) mRNA levels, were tested by qRT-PCR assay. Data were presented as mean ± SD of three independent experiments. ***P < 0.001; **P < 0.01
Fig. 6 Inhibition of miR-132-3p abrogated the progression of interstitial lung disease via targeting SPRY1 in NHLF. A The knockdown efficiency of SPRY1 in NHLF was tested by qRT-PCR and Western blot assays. B Cell viabilities were assessed in NHLF transfected with miR-NC, miR-132-3p inhibitor, miR-132-3p inhibitor + sh-NC, and miR-132-3p inhibitor + sh-SPRY1. C Cell colony numbers were detected in all groups. D Flow cytometry analyzed the apoptosis rate in all groups. E The anti-/pro-apoptosis protein expressions were assessed by Western blot assay. F-G The fibrosis/collagen-associated genes (α-SMA, TGF-β1, COL1A1, and COL3A1), and many pro-inflammatory mediators (IL-1β/6/8) mRNA levels, were tested by qRT-PCR assay. Data were presented as mean ± SD of three independent experiments. ***P < 0.001; **P < 0.01CC BY · PubMed Central

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Metadata

Authors
Fang, S., Wang, T., Weng, L., Han, X., Zheng, R., & Zhang, H.
Venue
World Journal of Surgical Oncology
Publisher
Springer Science and Business Media LLC
Published
2023-07-15
Volume
21
Issue
1
ISSN
1477-7819
DOI
10.1186/s12957-023-03095-6
PMC
PMC10349414
Cited by
13
References
34
Language
en
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