Neuropilin 1 modulates TGF‑β1‑induced epithelial‑mesenchymal transition in non‑small cell lung cancer

Abstract

Previously, the authors reported that neuropilin‑1 (NRP1) was significantly increased and acted as a vital promoter in the metastasis of non‑small cell lung cancer (NSCLC). However, the regulatory mechanism of NRP1 in NSCLC cell migration and invasion remained unclear. The present study aimed to explore the regulatory mechanism of NRP1 in the transforming growth factor‑β (TGF‑β) 1‑induced migration and invasion of NSCLC cells. The expression level of NRP1 was determined by RT‑qPCR analysis in human tissue samples with or without lymph node metastasis. Transwell assay and wound healing assay were conducted to determine the cell migration. Lentivirus‑mediated stable knockdown and overexpression of NRP1 cell lines were constructed. Exogenous TGF‑β1 stimulation, SIS3 treatment, western blot analysis and in vivo metastatic model were utilized to clarify the underlying regulatory mechanisms. The results demonstrated that the expression of NRP1 was increased in metastatic NSCLC tissues. NRP1 promoted NSCLC metastasis in vitro and in vivo. The Transwell assays, wound healing assays and western blot analysis revealed that the knockdown of NRP1 significantly inhibited TGF‑β1‑mediated EMT and migratory and invasive capabilities of NSCLC. Furthermore, the overexpression of NRP1 weakened the inhibitory effect of SIS3 on the NSCLC migration and invasion. Co‑IP assay revealed that NRP1 interacted with TGFβRII to induce EMT. On the whole, the findings of this study demonstrated that NRP1 was overexpressed in metastatic NSCLC tissues. NRP1 could contributes to TGF‑β1‑induced EMT and metastasis in NSCLC by binding with TGFβRII.

Figure 1

Knockdown of NRP1 suppresses NSCLC cell migration and invasion. (A) The protein expression levels of Snail, N-cadherin, Vimentin, MMP2 and MMP9 in stable NRP1-silenced A549 and H226 cells were detected by western blot analysis. (B) The migratory ability of stable A549 and H226 cells in which NRP1 was knocked down was investigated by wound healing assays (magnification, x200). (C) The migration and invasion of stable NRP1-silenced A549 and H226 cells were investigated by Transwell assays (magnification, x200). Data are shown as the means ± SD. Significantly different from the control (sh-NC) (^**P<0.01 and ^***P<0.001). NRP1, neuropilin 1; NSCLC, non-small cell lung cancer.

Figure 2

Overexpression of NRP1 promotes NSCLC cell migration and invasion. (A) The protein expression levels of Snail, N-cadherin, Vimentin, MMP2 and MMP9 in stable NRP1-overexpressed A549 and H226 cells were detected by western blot analysis. (B) The migratory ability of stable NRP1-overexpressing A549 and H226 cells was investigated by wound healing assays (magnification, x200). (C) The migration and invasion of stable NRP1-overexpressed A549 and H226 cells were investigated by Transwell assays (magnification, x200). Data are shown as the means ± SD. Significantly different from control (PVLX) (^**P<0.01 and ^***P<0.001). NRP1, neuropilin 1; NSCLC, non-small cell lung cancer.

Figure 3

High expression of NRP1 promotes NSCLC metastasis in vivo and is associated with TGFβR. (A) Surgically resected mouse lung tissues were fixed in Bouin's fluid. The pulmonary metastatic nodules on the surface of the lung tissue were counted (largest size was 1 mm), and the pulmonary micrometastases were detected by hematoxylin and eosin (H&E) staining; red arrowheads indicate micrometastases (magnification, x100). (B) A comparison of the number of pulmonary metastatic nodules between the sh-NRP1 and sh-NC groups. (C) Comparison of the relative mRNA expression of NRP1 detected by RT-qPCR between the tissues of NSCLC non-lymph node and lymph node metastasis. An unpaired t-test was used and the results were presented as means ± SD. Significantly different from the control (sh-NC or non-lymph node metastasis) (^*P<0.05 and ^***P<0.001). NRP1, neuropilin 1; NSCLC, non-small cell lung cancer; TGFβR, transforming growth factor-β receptor. (D-G) Correlation of NRP1 expression and SNAI1, SNAI2, MMP2 and TGFBR2 in linkedomics cohort (Pearson's correlation coefficient). (H) Interaction between NRP1 and TGFβRII was verified by co-immunoprecipitation assay. NRP1, neuropilin 1; NSCLC, non-small cell lung cancer; TGFβR, transforming growth factor-β receptor.

Figure 3

High expression of NRP1 promotes NSCLC metastasis in vivo and is associated with TGFβR. (A) Surgically resected mouse lung tissues were fixed in Bouin's fluid. The pulmonary metastatic nodules on the surface of the lung tissue were counted (largest size was 1 mm), and the pulmonary micrometastases were detected by hematoxylin and eosin (H&E) staining; red arrowheads indicate micrometastases (magnification, x100). (B) A comparison of the number of pulmonary metastatic nodules between the sh-NRP1 and sh-NC groups. (C) Comparison of the relative mRNA expression of NRP1 detected by RT-qPCR between the tissues of NSCLC non-lymph node and lymph node metastasis. An unpaired t-test was used and the results were presented as means ± SD. Significantly different from the control (sh-NC or non-lymph node metastasis) (^*P<0.05 and ^***P<0.001). NRP1, neuropilin 1; NSCLC, non-small cell lung cancer; TGFβR, transforming growth factor-β receptor. (D-G) Correlation of NRP1 expression and SNAI1, SNAI2, MMP2 and TGFBR2 in linkedomics cohort (Pearson's correlation coefficient). (H) Interaction between NRP1 and TGFβRII was verified by co-immunoprecipitation assay. NRP1, neuropilin 1; NSCLC, non-small cell lung cancer; TGFβR, transforming growth factor-β receptor.

Figure 4

Knockdown of NRP1 suppresses the TGF-β1-induced cell migration and invasion of NSCLC cells. (A and B) Following serum starvation for 24 h, stable NRP1-silenced A549 and H226 cells were treated with or without TGF-β1 (5 ng/ml) for 48 h. The expression of p-Smad3, Smad3, Snail, MMP2, MMP9, N-cadherin, Vimentin was analyzed by western blot analysis. (C) Stable NRP1-silenced A549 and H226 cells were treated with or without TGF-β (5 ng/ml) for 48 h, respectively, and the migratory ability of the cells was then investigated by wound healing assays. NRP1, neuropilin 1; NSCLC, non-small cell lung cancer; TGF-β, transforming growth factor-β. (D and E) Stable NRP1-silenced A549 and H226 cells were treated with or without TGF-β (5 ng/ml) for 48 h, respectively, and allowed to migrate through 8-µM pore Transwell inserts. The migrated cells were stained and counted in at least three microscopic fields (magnification, x100). The cells were allowed to invade through Matrigel-coated Transwell membranes, and invasive cells were stained and counted under a light microscope. Data are shown as the means ± SD. Significantly different from the control (^*P<0.05 and ^***P<0.001). NRP1, neuropilin 1; NSCLC, non-small cell lung cancer; TGF-β, transforming growth factor-β.

Figure 4

Knockdown of NRP1 suppresses the TGF-β1-induced cell migration and invasion of NSCLC cells. (A and B) Following serum starvation for 24 h, stable NRP1-silenced A549 and H226 cells were treated with or without TGF-β1 (5 ng/ml) for 48 h. The expression of p-Smad3, Smad3, Snail, MMP2, MMP9, N-cadherin, Vimentin was analyzed by western blot analysis. (C) Stable NRP1-silenced A549 and H226 cells were treated with or without TGF-β (5 ng/ml) for 48 h, respectively, and the migratory ability of the cells was then investigated by wound healing assays. NRP1, neuropilin 1; NSCLC, non-small cell lung cancer; TGF-β, transforming growth factor-β. (D and E) Stable NRP1-silenced A549 and H226 cells were treated with or without TGF-β (5 ng/ml) for 48 h, respectively, and allowed to migrate through 8-µM pore Transwell inserts. The migrated cells were stained and counted in at least three microscopic fields (magnification, x100). The cells were allowed to invade through Matrigel-coated Transwell membranes, and invasive cells were stained and counted under a light microscope. Data are shown as the means ± SD. Significantly different from the control (^*P<0.05 and ^***P<0.001). NRP1, neuropilin 1; NSCLC, non-small cell lung cancer; TGF-β, transforming growth factor-β.

Figure 5

Overexpression of NRP1 suppresses the SIS3-induced decrease in the migration and invasion of NSCLC cells. (A and B) Following serum starvation for 24 h, stable NRP1-silenced A549 and H226 cells were treated with or without SIS3 (3 µM) for 48 h. The expression of p-Smad3, Smad3, Snail, MMP2, MMP9, N-cadherin, Vimentin was analyzed by western blot analysis. (C) Stable NRP1-silenced A549 and H226 cells were treated with or without SIS3 (3 µM) for 48 h, respectively, and the migratory ability of the cells was then investigated by wound healing assays. NRP1, neuropilin 1; NSCLC, non-small cell lung cancer. (D and E) Stable NRP1-silenced A549 and H226 cells were treated with or without SIS3 (3 µM) for 48 h, respectively, and allowed to migrate through 8-µM pore Transwell inserts. The migrated cells were stained and counted in at least three microscopic fields (magnification, x100). The cells were allowed to invade through Matrigel-coated Transwell membranes, and invasive cells were stained and counted under a light microscope. Data are shown as the means ± SD. Significantly different from the control (PVLX) (^**P<0.01 and ^***P<0.001). NRP1, neuropilin 1; NSCLC, non-small cell lung cancer.

Figure 5

Overexpression of NRP1 suppresses the SIS3-induced decrease in the migration and invasion of NSCLC cells. (A and B) Following serum starvation for 24 h, stable NRP1-silenced A549 and H226 cells were treated with or without SIS3 (3 µM) for 48 h. The expression of p-Smad3, Smad3, Snail, MMP2, MMP9, N-cadherin, Vimentin was analyzed by western blot analysis. (C) Stable NRP1-silenced A549 and H226 cells were treated with or without SIS3 (3 µM) for 48 h, respectively, and the migratory ability of the cells was then investigated by wound healing assays. NRP1, neuropilin 1; NSCLC, non-small cell lung cancer. (D and E) Stable NRP1-silenced A549 and H226 cells were treated with or without SIS3 (3 µM) for 48 h, respectively, and allowed to migrate through 8-µM pore Transwell inserts. The migrated cells were stained and counted in at least three microscopic fields (magnification, x100). The cells were allowed to invade through Matrigel-coated Transwell membranes, and invasive cells were stained and counted under a light microscope. Data are shown as the means ± SD. Significantly different from the control (PVLX) (^**P<0.01 and ^***P<0.001). NRP1, neuropilin 1; NSCLC, non-small cell lung cancer.

Figure 6

A working model of the mechanistic interaction of NRP1 and TGF-β1-induced EMT involved in NSCLC metastasis.