Effects of EHD2 interference on migration of esophageal squamous cell carcinoma

Abstract

C-Terminal EH domain-containing protein 2 (EHD2) of the EHD family is associated with plasma membrane. We investigated the expression of EHD2 in human esophageal squamous cell carcinoma (ESCC) and the EHD2 expression to study the therapeutic effect of chemotherapy drugs. Western blot and immunohistochemistry were used to measure the expression of EHD2 protein in ESCC and adjacent normal tissue in 98 patients. EHD2 protein level was reduced in ESCC tissues in comparison with adjacent normal tissues. Under-expression of EHD2 increased the motility property of ESCC cell TE1 in vitro by wound-healing assays and transwell migration assays, and it was concurrent with the decreased expression of epithelial marker E-cadherin. Under-expression of EHD2 in TE1 can cause resistance to cisplatin. Our results suggested that EHD2 low expression is involved in the pathogenesis of ESCC, and it might be a favorable independent poor prognostic parameter for ESCC.

Fig. 1

Immunohistochemical staining reveals EHD2 and E-cadherin expression in paraffin-embedded ESCC tissues. a, b, e, f Cancer tissues with no lymph node metastasis showed high EHD2 and E-cadherin expression (×200 and ×400). c, d, g, h Cancer tissues with lymph nodes metastases showed low EHD2 and E-cadherin expression (×200 and ×400). Details of the experiments are given in “Materials and methods”

Fig. 2

Expression of EHD2 in human ESCC. a Expression of EHD2 in eight representative paired samples of esophageal tumor tissues (T) and adjacent normal tissues (N). b The bar chart demonstrates the ratio of EHD2 protein to GAPDH for the above by densitometry. The data are mean ± SEM (*P < 0.01, compared with adjacent tumor tissues)

Fig. 3

Cumulative survival curves according to EHD2 expression. On the basis of score of EHD2, patients were divided into high EHD2 expressers (score ≥ 2) and low EHD2 expressers (score < 2). Patients in the low-expression EHD2 group had significantly shorter overall survival

Fig. 4

Under-expression of EHD2 facilitates the migration and invasion of TE1 cells. Migration and invasion of cells treated with siEHD2 (or negative control) were analyzed at 48 h post-infection. a Wound-healing assay. Photographs represented the cells migrated into the wounded area and b histogram showed the relative migration distance of cells. The data are mean ± SEM (n = 3, ^{#,^,}*P < 0.01, compared with 0 h). c Transwell assay. Photographs represented the cells travelled through the micropore membrane and d histogram showed the percentage of migrant cells. The data are mean ± SEM (n = 3, *P < 0.01, compared with Mock). e EHD2 interference efficiency was certified by Western blot. Data represent mean ± SEM from four independent experiments; P < 0.01 by t test

Fig. 5

Interference of EHD2 affected TE1 cells proliferation and was insensitive to chemotherapy drugs. a Treat TE1 cells with cisplatin for 48 h at 5, 10, 20 μmol/L, respectively, after interference of EHD2 for 48 h. The data are mean ± SEM (n = 3, *P < 0.01, compared with Mock). b Western blot analyses proliferation of TE1 cells when interference of EHD2 and with or without treatment of ciaplatin