Oncogene 5'-3' exoribonuclease 2 enhances epidermal growth factor receptor signaling pathway to promote epithelial-mesenchymal transition and metastasis in non-small-cell lung cancer

Abstract

Objective: Epithelial-mesenchymal transition (EMT) and metastasis are the primary causes of mortality in non-small-cell lung cancer (NSCLC). 5'-3' exoribonuclease 2 (XRN2) plays an important role in the process of tumor EMT. Thus, this investigation mainly aimed to clarify the precise molecular pathways through which XRN2 contributes to EMT and metastasis in NSCLC.

Material and methods: Western blot and quantitative real-time polymerase chain reaction were first used to assess XRN2 levels in NSCLC cells. Subsequently, short hairpin RNA-XRN2 (Sh-XRN2) and XRN2 overexpression (Ov-XRN2) plasmids were transfected to NSCLC cells. The effects of Sh-XRN2 and Ov-XRN2 on NSCLC cell migration and invasion were evaluated by Transwell assay. Western blot experiments were conducted to assess the effects of Sh-XRN2 and Ov-XRN2 on proteins related to EMT and the epidermal growth factor receptor (EGFR) signaling pathway in H460 cells. Then, Sh-XRN2 and EGFR overexpression (Ov-EGFR) plasmids were transfected to NSCLC cells. Changes in NSCLC cell migration and invasion were measured using a Transwell assay with Sh-XRN2 and Sh-XRN2+Ov-EGFR. Changes in the expression of proteins related to EMT in NSCLC cells were detected by Western blot assays with Sh-XRN2 and Sh-XRN2+Ov-EGFR. Furthermore, a subcutaneous tumor model for NSCLC was established. Immunohistochemical analysis was performed to assess the levels of Cluster of Differentiation 31 (CD31) in lung metastatic lesions. H460 cells transfected with Sh-XRN2, Ov-XRN2 or Sh-XRN2+Ov-EGFR were co-cultured with human umbilical vein endothelial cells (HUVECs) to assess the tube formation ability of the cells.

Results: Compared with those observed in human bronchial epithelial cells (BEAS-2B cells), XRN2 expression levels were significantly upregulated in NSCLC cell lines (H460 cells) (P < 0.001). XRN2 overexpression considerably promoted the NSCLC cell migration and invasion, EMT process, and tube formation ability of HUVECs (P < 0.001). On the contrary, XRN2 knockdown led to a reduction in these processes. In addition, XRN2 overexpression increased the expression levels of CD31 in lung metastatic lesions and activated the phosphorylation of EGFR signaling pathway (P < 0.001). Furthermore, Sh-XRN2+Ov-EGFR significantly promoted migration, invasion, and EMT processes in H460 cells (P < 0.001). In the meantime, compared with the co-H460+Sh-XRN2+Ov-NC group, co-H460+Sh-XRN2+Ov-EGFR significantly enhanced the tube formation ability of HUVECs (P < 0.001).

Conclusion: XRN2 promoted EMT and metastasis in NSCLC through improving the phosphorylation of the EGFR signaling pathway in NSCLC cells.

Keywords: 5’-3’ exoribonuclease 2; epidermal growth factor receptor; epithelial– mesenchymal transition; metastasis; non-small-cell lung cancer.

Figure 1:

XRN2 was upregulated in NSCLC. (a) The messenger RNA levels of XRN2 in the H460 NSCLC cell line and BEAS-2B human bronchial epithelial cells. (b and c) Protein expression levels of XRN2 in H460 and BEAS-2B cells. n = 6. (***P < 0.001). (XRN2: 5’-3’ exoribonuclease 2, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, H460 cells: NSCLC cell line; BEAS-2B: Human bronchial epithelial cells, NSCLC: Non-small-cell lung cancer.)

Figure 2:

XRN2 promoted migration and EMT progression in NSCLC cells. (a-c) Validation of XRN2 overexpression and knockdown efficiency in H460 cells. (d-g) Migration and invasion assay of H460 cells after XRN2 overexpression and knockdown. (h-k) Protein levels of E-cadherin, N-cadherin, and vimentin in H460 cells after XRN2 overexpression and knockdown. n = 6. (**P < 0.01 and ***P < 0.001). (Ov-NC: Overexpress negative control, XRN2: 5’-3’ exoribonuclease 2, Sh-NC: ShRNA negative control, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, H460 cells: NSCLC cell line, BEAS-2B: Human bronchial epithelial cells, NSCLC: Non-small-cell lung cancer.)

Figure 3:

XRN2 promoted angiogenesis in NSCLC lung metastasis. (a and b) HE stained images of lung metastases. (c and d) IHC analysis of CD31 + cells in lung metastatic lesions. (e and f) Images of tube formation by HUVECs co-cultured with H460-Ov-XRN2 or H460-Sh-XRN2 cells. (g-i) mRNA and protein expression levels of VEGFA in HUVECs under co-culture conditions. n = 6. (**P < 0.01; ***P < 0.001). (Ov-NC: Overexpress negative control, XRN2: 5’-3’ exoribonuclease 2, Sh-NC: ShRNA negative control, CD31: Cluster of differentiation 31, FOV: Field of view, VEGFA: Vascular endothelial growth factor A, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, H460 cells: NSCLC cell line, BEAS-2B: Human bronchial epithelial cells, NSCLC: Non-small-cell lung cancer, Ov-XRN2: XRN2 overexpression, HUVECs: Human umbilical vein endothelial cells .)

Figure 4:

XRN2 overexpression promoted the phosphorylation of EGFR in NSCLC cells. (a) The protein bands of p-EGFR and EGFR. (b) Relative expression levels of the p-EGFR/EGFR protein ratio. (c) The EGFR mRNA levels in H460 cells. n = 6. (***P < 0.001). (Ov-NC: Overexpress negative control, XRN2: 5’-3’ exoribonuclease 2, Sh-NC: ShRNA negative control, EGFR: Epidermal growth factor receptor, p-EGFR: Phosphorylation epidermal growth factor receptor, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, NSCLC: Non-small-cell lung cancer.)

Figure 5:

EGFR mediated the biological functions of XRN2 in NSCLC metastasis. (a-d) Transwell assays of H460 cells after transfection with Sh-XRN2 and Ov-EGFR. (e-h) The protein expression levels of E-cadherin, N-cadherin, and vimentin in H460 cells post-transfected with Sh-XRN2 and Ov-EGFR. (i and j) Tube formation by HUVECs co-cultured with H460-Sh-XRN2 or H460-Sh-XRN2+Ov-EGFR. n = 6. (*P < 0.05, **P < 0.01 and ***P < 0.001). (XRN2: 5’-3’ exoribonuclease 2, Sh-NC: ShRNA negative control, Ov-NC: overexpression negative control, EGFR: Epidermal growth factor receptor, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, NSCLC: Non-small cell lung cancer, Ov-EGFR: EGFR overexpression, HUVECs: Human umbilical vein endothelial cells.)