Over-expression of Nectin-4 promotes progression of esophageal cancer and correlates with poor prognosis of the patients

Fig. 1. Nectin-4 expression profiles in normal human tissues. a, b The RNA (a) and protein (b) expression profiles of Nectin-4 in normal human tissues. Data credit: Human Protein Atlas. Data summary images were obtained from: https://www.proteinatlas.org/ENSG00000143217-NECTIN4/tissue

Fig. 2. Survey of Nectin-4 expression at mRNA level in human EC tissues based on TCGA data. a, b Based on the TCGA data from http://gepia.cancer-pku.cn/, we could not find any significant difference of Nectin-1 and Nectin-2 at mRNA level between cancer tissues and adjacent normal tissues. c, d We found that the mRNA expression level of Nectin-3 was significantly higher in cancer tissues compared with that in adjacent normal tissues (P < 0.05), and the mRNA expression level of Nectin-4 was significantly higher in adjacent normal tissues compared with cancer tissues (P < 0.05). e–g We found that the expression of Nectin-4 at the mRNA level in EC tissues was positively and significantly correlated with that of Nectin-1 (P < 0.0001), while it was negatively and significantly correlated with that of Nectin-2 (P < 0.0001) and Nectin-3 (P < 0.0001). h, i We didn’t find any significant correlations between patients’ postoperative survival and Nectin-1 or Nectin-2. j The overall survival rate of the patients with higher expression of Nectin-3 at the mRNA level was significantly greater compared with those showing lower Nectin-3 expression (P = 0.006). k The patients with lower expression of…

Fig. 3. Nectin-4 expression in EC tissues. a, b Positive staining of Nectin-4 could be found in the cytoplasm and on the membrane of cancer cells, while weak or negative staining of Nectin-4 was found in normal esophageal tissues. c The staining intensity of Nectin-4 in EC tissues was significantly higher than that in adjacent normal tissues (P < 0.0001). d The survival analysis showed that the overall survival rate of the patients with higher Nectin-4 expression was significantly poorer compared with those showing lower Nectin-4 expression (HR = 1.704, 95% CI 1.027–2.825, P = 0.039)

Fig. 4. Knockdown expression and over-expression of Nectin-4 in human EC cell lines Eca-109 and TE-1. a The knockdown expression of Nectin-4 at mRNA level by using RNAi approach in human EC lines was confirmed by real-time RT-PCR, and the Nectin-4 mRNA expression level in LV-Nectin-4-shRNA group cells was significantly lower than that in LV-NC group cells (both in Eca-109 and in TE-1, P < 0.0001). b The decreased Nectin-4 protein expression after knockdown in human EC cell lines Eca-109 and TE-1, were confirmed by using western blot method. c The Nectin-4 protein expression level in LV-Nectin-4-shRNA group cells was significantly lower than that in LV-NC group cells (in Eca-109, P < 0.01, and in TE-1, P < 0.001). d The over-expression of Nectin-4 at mRNA level in human EC lines was confirmed by real-time RT-PCR, which showed that the increased Nectin-4 mRNA expression level in LV-Nectin-4-OE group cells compared with LV-Vector-Ctrl group cells (both in Eca-109 and in TE-1, P < 0.0001). e The increased Nectin-4 protein expression in human EC cell lines Eca-109 and TE-1 were confirmed by using western blot method. f The Nectin-4 protein expression level in LV-Nectin-4-OE group cells was…

Fig. 5. Effect of intervention of Nectin-4 expression on cell proliferation of human EC cell lines. We examined the intervention of Nectin-4 expression on the cell proliferation in vitro using CCK-8 assay in human EC cell lines. a In Eca-109 cells, at 48 and 72 h after seeding, the cell proliferation rate of LV-Nectin-4-shRNA group was significantly lower compared with the LV-NC group (both P < 0.01). b In Eca-109 cells, at 48 and 72 h after seeding, the cell proliferation rate of LV-Nectin-4-OE group was significantly higher compared with the LV-Vector-Ctrl group (both P < 0.01). c In TE-1 cells, at 24, 48 and 72 h after seeding, the cell proliferation rate of LV-Nectin-4-shRNA group was significantly lower compared with the LV-NC group (all P < 0.01). d In TE-1 cells, at 48 and 72 h after seeding, the cell proliferation rate of LV-Nectin-4-OE group was significantly higher compared with the LV-Vector-Ctrl group (both P < 0.01)

Fig. 6. The intervention of Nectin-4 expression affects the migration ability of human EC cell lines. The wound healing assay was performed to evaluate the effect of the intervention of Nectin-4 expression on the migration ability of human EC cell lines. a Knockdown of Nectin-4 expression significantly decreased the cell migration ability of Eca-109 as well TE-1 cells, the cell-free area of LV-Nectin-4-shRNA group was significantly wider than that of LV-NC group at 24 h (in Eca-109, P < 0.05, and in TE-1, P < 0.01). b Over-expression of Nectin-4 expression significantly increased the cell migration ability of Eca-109 as well TE-1 cells, the cell-free area of LV-Nectin-4-OE group was significantly narrower than that of LV-Vector-Ctrl group at 24 h (both P < 0.01)

Fig. 7. The intervention of Nectin-4 expression affects the invasion ability of human EC cell lines. The transwell invasion assay was performed to evaluate the effect of the intervention of Nectin-4 expression on the invasion ability of human EC cell lines. a Knockdown of Nectin-4 expression significantly decreased the number of invaded cells stained with Cristal Violet in LV-Nectin-4-shRNA group cells compared with LV-NC group cells (in Eca-109, P < 0.01, and in TE-1, P < 0.001). b Nectin-4 over-expression significantly increased the number of invaded cells stained with Cristal Violet in LV-Nectin-4-OE group cells compared with LV-Vector-Ctrl group cells (both P < 0.001)

Fig. 8. Intervention of Nectin-4 expression effects the tumor growth in the subcutaneous transplantation mouse model. The subcutaneous transplantation mouse model was used to evaluate the effect of the intervention of Nectin-4 expression on the tumor growth of human EC cell lines. The Eca-109 or TE-1 cells from LV-NC, LV-Nectin-4-shRNA, LV-Vector-Ctrl and LV-Nectin-4-OE groups were injected subcutaneously into nude mice (n = 5, respectively). a In tumor models established by using Eca-109 cells, the tumor growth curves showed that knockdown of Nectin-4 expression significantly inhibits tumor growth (P < 0.0001), and the Nectin-4 over-expression significantly promotes tumor growth (P < 0.0001). b The tumor tissues resected from the tumor models established by Eca-109 cells from each groups were listed. c After 28 days, in tumor models established by using Eca-109 cells, the tumor weight of LV-Nectin-4-shRNA group was lighter than that of LV-NC group (P < 0.05), the tumor weight of LV-Nectin-4-OE group trended heavier than that of LV-Vector-Ctrl group (P < 0.001). d In tumor models established by using TE-1 cells, the tumor growth curves showed that knockdown of Nectin-4 expression…