The effects of vascular endothelial growth factor C knockdown in esophageal squamous cell carcinoma

Abstract

Purpose: We investigated the role of vascular endothelial growth factor C (VEGF-C) in esophageal squamous cell carcinoma (ESCC) by knocking down VEGF-C expression in the ESCC cell line EC9706.

Methods: Immunohistochemistry and in situ hybridization techniques were used to detect the expression of VEGF-C expression in ESCC tissues. We also investigated the relationship between VEGF-C expression and lymph node metastasis. We designed a siRNA expression plasmid for VEGF-C and transfected it into EC9706 cells. Stable clones were selected, and VEGF-C expression was analyzed by RT-PCR and western blotting. Cells were inoculated into nude mice. The expression of VEGF-C in the resulting tumors was analyzed by immunohistochemistry and in situ hybridization.

Results: VEGF-C is highly expressed in ESCC and correlated with lymph node metastasis, as high levels were observed in patients presenting with lymph node metastases relative to those who did not (P < 0.01). Transfection with VEGF-C-siRNA decreased the expression of VEGF-C mRNA and protein. ESCC cells stably transfected with VEGF-C-siRNA expressed very low levels of VEGF-C (P < 0.01 compared with control). This knockdown effect persisted when the cells were inoculated into nude mice and allowed to form tumors.

Conclusions: The siRNA-targeted knockdown of VEGF-C led to a significant reduction in VEGF-C expression. This siRNA technique could be used for gene therapy in ESCC.

Fig. 1

VEGF-C expression in ESCC. a VEGF-C protein expressed in the cytoplasm of tumor cells. Immunohistochemistry, ×100 magnification. b VEGF-C mRNA expressed in the cytoplasm of tumor cells. In situ hybridization, BCIP/NB, ×100 magnification

Fig. 2

EC9706 cells transfected with VEGF-C siRNA showed decreased VEGF-C protein levels. Note: 1 untransfected control, 2 transfected with scramble plasmid, 3–5 transfected with siRNA plasmid

Fig. 3

siRNA inhibited VEGF-C mRNA expression in EC9706 cells. Note: M DNA marker, 1 control EC9706 cells, 2 scrambled siRNA-transfected EC9706 cells, 3, 4, 5 VEGF-C siRNA-transfected EC9706 cells

Fig. 4

VEGF-C protein expression in mouse tumors. a Tumors arising from untransfected control EC9706 cells. b Tumors from scrambled siRNA-transfected EC9706 cells. c VEGF-C siRNA-transfected EC9706 cells. Immunohistochemistry, ×400 magnification

Fig. 5

VEGF-C mRNA expression in mouse tumors. a Tumors arising from untransfected control EC9706 cells. b Tumors from scrambled siRNA-transfected EC9706 cells. c Tumors from siRNA-transfected EC9706 cells. In situ hybridization, BCIP/NBT. ×400 magnification