Thrombospondin-1 and microRNA-1 expression in response to multiwalled carbon nanotubes in alveolar epithelial cells

Abstract

Thrombospondin-1 (TSP-1) is a glycoprotein that plays a role in extracellular matrix (ECM) remodeling. Previously, we have shown that multiwalled carbon nanotubes (MWCNT) regulate ECM components TGFβ and its target Col3A1 in alveolar epithelial cells. In this study, we investigated the effect of MWCNT on TSP-1 and microRNA-1 (miR-1) in the regulation of TGFβ in ECM remodeling using alveolar epithelial A549 cells. A549 cells were treated with MWCNT (20 or 50 µg/mL) for 6 or 24 h and the expression of TSP-1 and miR-1, and the exogenous miR-1 effect on cell morphology were analyzed. MWCNT induced in a time- and dose-dependent manner the expression of TSP-1. miR-1 was suppressed by MWCNT after 6 or 24 h of treatment regardless of the dose. TSP-1 and miR-1 negatively correlated with each other, r = -0.58. Exogenous administration of miR-1 induced alveolar epithelial cell morphology changes including cell clustering, whereas inhibition of miR-1 induced less cell to cell contact, cell rounding, and cellular projections. IntAct molecular network interactions analysis revealed that TSP-1 interacts with 21 molecular factors including ECM genes, and molecules. These results indicate a relationship between that TSP-1, MWCNT, and TGFβ, and suggest TSP-1 may play a role in MWCNT-induced TGFβ and ECM remodeling. Moreover, these data also suggest an inverse relationship between TSP-1 and miR-1 and a potential role of miR-1 in MWCNT-induced fibrotic signaling. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1596-1606, 2017.

Keywords: Alveolar epithelial cells; ECM; MWCNT; Pulmonary fibrosis; TSP-1; miR-1.

Fig. 1

A) Bioinformatics prediction model for miR-1 and its target gene associated with lung fibrosis/remodeling and assay for miR-1 and target gene. B) 3′- UTR sequence of miR-1 putative binding sites to target gene TSP-1 is given in the 5′- to 3′- orientation.

Fig. 2

TSP-1 regulation by MWCNT. A) TSP-1 mRNA level in the cells treated with MWCNT (20 or 50 µg/ml) for 6 or 24 h. The data is presented as mRNA fold change relative to 18S. The data represents the mean ± SEM, n=3. B) TSP-1 immunohistochemistry detected using TSP-1 immunofluorescence (green) and DAPI (nucleus). The arrow points to immunofluorescence of TSP-1. C) Western blot analysis of TSP-1 and beta-actin in A549 cells treated with MWCNT as indicated in the figure. One representative blot is shown. D) TSP-1 intensity analysis normalized to beta actin and relative to control untreated samples. The data represents the mean ± SEM, n=3. *Statistically significant versus control, ANOVA, p ≤ 0.05.

Fig. 2

TSP-1 regulation by MWCNT. A) TSP-1 mRNA level in the cells treated with MWCNT (20 or 50 µg/ml) for 6 or 24 h. The data is presented as mRNA fold change relative to 18S. The data represents the mean ± SEM, n=3. B) TSP-1 immunohistochemistry detected using TSP-1 immunofluorescence (green) and DAPI (nucleus). The arrow points to immunofluorescence of TSP-1. C) Western blot analysis of TSP-1 and beta-actin in A549 cells treated with MWCNT as indicated in the figure. One representative blot is shown. D) TSP-1 intensity analysis normalized to beta actin and relative to control untreated samples. The data represents the mean ± SEM, n=3. *Statistically significant versus control, ANOVA, p ≤ 0.05.

Fig. 2

TSP-1 regulation by MWCNT. A) TSP-1 mRNA level in the cells treated with MWCNT (20 or 50 µg/ml) for 6 or 24 h. The data is presented as mRNA fold change relative to 18S. The data represents the mean ± SEM, n=3. B) TSP-1 immunohistochemistry detected using TSP-1 immunofluorescence (green) and DAPI (nucleus). The arrow points to immunofluorescence of TSP-1. C) Western blot analysis of TSP-1 and beta-actin in A549 cells treated with MWCNT as indicated in the figure. One representative blot is shown. D) TSP-1 intensity analysis normalized to beta actin and relative to control untreated samples. The data represents the mean ± SEM, n=3. *Statistically significant versus control, ANOVA, p ≤ 0.05.

Fig. 2

TSP-1 regulation by MWCNT. A) TSP-1 mRNA level in the cells treated with MWCNT (20 or 50 µg/ml) for 6 or 24 h. The data is presented as mRNA fold change relative to 18S. The data represents the mean ± SEM, n=3. B) TSP-1 immunohistochemistry detected using TSP-1 immunofluorescence (green) and DAPI (nucleus). The arrow points to immunofluorescence of TSP-1. C) Western blot analysis of TSP-1 and beta-actin in A549 cells treated with MWCNT as indicated in the figure. One representative blot is shown. D) TSP-1 intensity analysis normalized to beta actin and relative to control untreated samples. The data represents the mean ± SEM, n=3. *Statistically significant versus control, ANOVA, p ≤ 0.05.

Fig. 3

The effect of MWCTN on miR-1 level. The cells were treated with MWCNT, 20 or 50 µg/ml, for 6 and 24 h. MicroRNA was detected using real-time qPCR according to TaqMan assay as described in Materials and Methods. The data shown represents the mean ± SEM, n=3. *Statistically significant versus control, ANOVA, p ≤ 0.05.

Fig. 4

Correlation analysis between miR-1 and TSP-1 mRNA. The data were analyzed using Pearson correlation analysis between TSP-1 mRNA fold change and miR-1 fold change.

Fig. 5

Cell migration in wound healing after exposure to MWNT (20 or 50 µg/ml) for 24 h. A). The cell monolayer was wounded using wound scratch assay and cell migration toward wound healing was observed after 24 h. One representative micrograph is shown. B). Counted migrating cells toward wound healing. Data is presented as mean ± SEM, n=3. *Statistically significant versus control, ANOVA, p ≤ 0.05.

Fig. 5

Cell migration in wound healing after exposure to MWNT (20 or 50 µg/ml) for 24 h. A). The cell monolayer was wounded using wound scratch assay and cell migration toward wound healing was observed after 24 h. One representative micrograph is shown. B). Counted migrating cells toward wound healing. Data is presented as mean ± SEM, n=3. *Statistically significant versus control, ANOVA, p ≤ 0.05.

Fig. 6

Comparison of cell morphology after microRNA-1 overexpression or inhibition. A) Control; B) miR-1 mimic; C) miR-1 inhibitor. The cells were seeded in growth media for 24 h followed by treatments with miR-1 mimic and miR-1inhibitor using lipofectamine in basal media for 48 h. Representative micrographs of one experiment, n=3.

Fig. 7

Molecular network analysis of TSP-1 interactions with other molecular targets. The network was generated using IntAct. The map shows that TSP-1 interacts with 22 other genes and molecules of ECM including TGFβ and Col.