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. 2018 Oct;7(10):5118-5129.
doi: 10.1002/cam4.1758. Epub 2018 Sep 6.

TGF-β-mediated exosomal lnc-MMP2-2 regulates migration and invasion of lung cancer cells to the vasculature by promoting MMP2 expression

Dong-Ming Wu  1 , Shi-Hua Deng  1 , Teng Liu  1 , Rong Han  1 , Ting Zhang  1 , Ying Xu  1
Affiliations

TGF-β-mediated exosomal lnc-MMP2-2 regulates migration and invasion of lung cancer cells to the vasculature by promoting MMP2 expression

Dong-Ming Wu et al. Cancer Med. 2018 Oct.

Erratum in

  • Corrigendum.
    [No authors listed] [No authors listed] Cancer Med. 2020 Jul;9(13):4876. doi: 10.1002/cam4.3145. Epub 2020 Jun 14. Cancer Med. 2020. PMID: 32619337 Free PMC article. No abstract available.

Abstract

Previous studies indicated that transforming growth factor (TGF)-β-mediated exosomal microRNAs (miRNAs) regulate the migration and invasion of lung cancer cells; however, whether and how TGF-β-mediated exosomal long noncoding (lnc) RNAs regulate migration and invasion of lung cancer cells remains unclear. Here, coculture experiments showed that TGF-β pretreatment increased the migration and invasion potential of lung cancer cells and TGF-β pretreated A549 cells increases vascular permeability. Furthermore, we found that TGF-β-mediated exosomes, as carriers of intercellular communication, regulated lung cancer invasion, and vascular permeability. Transcriptional analysis also revealed that lnc-MMP2-2 was highly enriched in TGF-β-mediated exosomes and might function by increasing the expression of matrix metalloproteinase (MMP)2 through its enhancer activity, with ectopic expression and silencing of lnc-MMP2-2 affecting lung cancer invasion and vascular permeability. Additionally, lnc-MMP2-2 and MMP2 expression was assessed semiquantitatively, and tissue-specific correlations between lnc-MMP2-2 and MMP2 expression were evaluated. These results suggested that exosomal lnc-MMP2-2 might regulate the migration and invasion of lung cancer cells into the vasculature by promoting MMP2 expression, suggesting this lncRNA as a novel therapeutic target and predictive marker of tumor metastasis in lung cancer.

Keywords: TGF-β; exosomes; lncRNA; lung cancer; matrix metalloproteinase.

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Figures

Figure 1
Figure 1
TGF‐β pretreated A549 cells increase the migratory and invasive activity of lung cancer cells. A, Schematic representation of the coculture assay. B, Wound healing results for Ctl/A549 (Ctl) and TGF‐β/A549 (TGF‐β) cells. Scale bar, 500 μm. C, Quantification of wound healing. D, Quantification of transwell migration and matrigel invasion. E, Transwell migration and matrigel invasion by Ctl and TGF‐β A549 cells. Scale bar, 100 μm. F‐H, Expression of E‐cadherin, N‐cadherin, and vimentin in Ctl and TGF‐β A549 cells as measured by (F‐G) western blot and (H)immunofluorescence staining (scale bar, 50 μm). *P < 0.05; **P < 0.01 versus Ctl
Figure 2
Figure 2
TGF‐β pretreated A549 cells increase HMVEC‐L monolayers permeability and downregulate tight‐junction protein expression. A, Schematic representation of the co‐culture assay. B, Schematic representation of the transwell chamber used for assaying transport across an endothelial monolayer. C, The permeability of pre‐cocultured HMVEC‐L monolayers grown on 0.4‐μm filters as measured by the appearance of rhodamine B isothiocyanate‐dextran, which was added in the upper well at the beginning of the experiment and in the bottom well after a 1‐h incubation. D and E, Expression of occludin and zonula occludens‐1 in A549/HMVEC‐L(Ctl) and TGF‐β+A549/HMVEC‐L cells (TGF‐β) as measured by western blot(D) and immunofluorescence staining(E) (scale bar, 50 μm). **P < 0.01 versus Ctl
Figure 3
Figure 3
TGF‐β‐mediated exosome release promotes the migration and invasion of A549 cells and attenuates barrier functions at the HMVEC‐L monolayer. A, TEM images of exosomes secreted by A549 cells(exo) and TGF‐β‐pretreated A549 cells(Texo). B, exosome characteristic proteins Alix, CD9, CD63 were detected by western blot. C, Wound healing in exo and Texo treated A549 cells. Scale bar, 500 μm. D, Quantification of wound healing. E, Transwell migration and matrigel invasion by exo and Texo treated A549 cells. Scale bar, 100 μm. F, Quantification of transwell migration and matrigel invasion. G and H, Expression of E‐cadherin, N‐cadherin, and vimentin in exo and Texo treated A549 cells as measured by (G) immunofluorescence staining (scale bar, 50 μm) and (H) western blot. I and J, Expression of occludin and zonula occludens‐1 in exo and Texo treated HMVEC‐L cells as measured by (I) immunofluorescence staining (scale bar, 50 μm) and (J) western blot. K, Permeability of the exo and Texo treated HMVEC‐L monolayers. **P < 0.01; ***P < 0.001 versus exo
Figure 4
Figure 4
Lnc‐MMP2‐2 is highly enriched in TGF‐β‐mediated exosomes and alters MMP2 levels. A, Scatter Plots revealed differences in lncRNAs. B, Venn diagram revealed differences in lncRNAs. C, qRT‐PCR analysis of lnc‐MMP2‐2 levels in TGF‐β mediated A549 cell‐derived exosome (Texo) and Ctl exo(exo). D, FISH analysis of lnc‐MMP2‐2 levels in exosomes treated A549 cells. E, Gene location analysis shows that lnc‐MMP2‐2 is located in the upstream of MMP2, indicating its transcriptional enhancement potentiality. F and G, qRT‐PCR (F) and Western blot (G) analysis of the effects of the treatment of Texo in A549 cells on lnc‐MMP2‐2 and MMP2 expression. (G) Luciferase asssay was performed to evaluate the activity of the MMP2 promoter in A549 cells with lnc‐MMP2‐2 overexpression. I and J, qRT‐PCR (I) and Western blot (J) analyses of the effects of lnc‐MMP2‐2 overexpression on MMP2 expression. **P < 0.01; ***P < 0.001 versus exo
Figure 5
Figure 5
Overexpression of lnc‐MMP2‐2 promotes migration and invasion of A549 cells and attenuates barrier functions at the HMVEC‐L monolayer. A, Lnc‐MMP2‐2 expression in control (Vector) and lnc‐MMP2‐2‐transfected (lnc‐MMP2‐2) A549 and HMVEC‐L cells as measured by qRT‐PCR. B, Wound healing in Vector and lnc‐MMP2‐2 A549 cells. Scale bar, 500 μm. C, Quantification of wound healing. D, Transwell migration and matrigel invasion by Vector and lnc‐MMP2‐2 A549 cells. Scale bar, 100 μm. E, Quantification of transwell migration and matrigel invasion. F and G, Expression of E‐cadherin, N‐cadherin, and vimentin in Vector and lnc‐MMP2‐2 A549 cells as measured by (F) immunofluorescence staining (scale bar, 50 μm) and (G) western blot. H and I, Expression of occludin and zonula occludens‐1 in Vector and lnc‐MMP2‐2 HMVEC‐L cells as measured by (H) immunofluorescence staining (scale bar, 50 μm) and (I) western blot. J, Permeability of Vector and lnc‐MMP2‐2 HMVEC‐L monolayers. **P < 0.01; ***P < 0.001 versus Vector
Figure 6
Figure 6
Lnc‐MMP2‐2 silencing inhibits the migration and invasion of A549 cells and protects barrier functions at the HMVEC‐L monolayer. A, Lnc‐MMP2‐2 expression in control (shCtl) and knockdown (shlnc‐MMP2‐2) A549 and HMVEC‐L cells as measured by qRT‐PCR. B, Wound healing by shCtl and shlnc‐MMP2‐2 A549 cells. Scale bar, 500 μm. C, Quantification of wound healing. D, Transwell migration and matrigel invasion by shCtl and shlnc‐MMP2‐2 A549 cells. Scale bar, 100 μm. E, Quantification of transwell migration and matrigel invasion. F and G, Expression of E‐cadherin, N‐cadherin, and vimentin in shCtl and shlnc‐MMP2‐2 A549 cells as measured by (F) immunofluorescence staining (scale bar, 50 μm) and (G) western blot. H and I, Expression of occludin and zonula occludens‐1 in shCtl and shlnc‐MMP2‐2 HMVEC‐L cells as measured by (H) immunofluorescence staining (scale bar, 50 μm) and (I) western blot. J, Permeability of shCtl and shlnc‐MMP2‐2 HMVEC‐L monolayers. *P < 0.05; **P < 0.01 versus shCtl
Figure 7
Figure 7
Lnc‐MMP2‐2 is associated with MMP2 expression and metastatic progression in lung cancer. A, Representative images of MMP2 and lnc‐MMP2‐2 staining in normal, primary, and metastatic lung tissue. Scale bar, 50 μm. B, Immunohistochemistry scores for MMP2 in normal (n = 5), primary (n = 14), and metastatic (n = 4) lung tissue. C, Fluorescence in situ hybridization scores for lnc‐MMP2‐2 in normal (n = 5), primary (n = 14), and metastatic (n = 4) lung tissue. D, Correlation between MMP2 and lnc‐MMP2‐2 in all cases (n = 23). **P < 0.01; ***P < 0.001
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