Blocking effects of siRNA on VEGF expression in human colorectal cancer cells.

Abstract

Aim: To investigate the expression of vascular endothelial cell growth factor (VEGF) and its receptors Fms-like tyrosine kinase 1 (FLT-1) and fetal liver kinase 1 (FLK-1) in colorectal carcinoma (CRC), and the blocking effects of small interfering RNAs (siRNAs) on VEGF expression in human colorectal cancer HCT116 cells.

Methods: Immunohistochemical staining for VEGF, FLT-1 and FLK-1 proteins was performed in 82 cases of CRC and 14 normal colorectal mucosae. A siRNA targeting VEGF was synthesized and transfected into HCT116 cells using lipofectamine 2000. Immunocytochemical staining and Western blotting analyses were performed to detect the expression of VEGF protein. The suppressive effect of the siRNA on cell proliferation was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide (MTT) assay. Cellular apoptosis was detected using flow cytometry (FCM).

Results: The expression of VEGF, FLT-1 and FLK-1 in tumor tissues was significantly higher than that in normal tissues (P = 0.008, P = 0.000, P = 0.000). The expression of VEGF was positively correlated with both lymph node metastasis and clinical stage (P = 0.009 and P = 0.025, respectively). Immunocytochemistry showed that the expression of VEGF was weakly positive and Western blotting indicated a significant reduction in VEGF-siRNA cell protein levels. VEGF-siRNA cell growth inhibition was assessed by the MTT assay, and the tumor cell proliferation rate was significantly different at 24, 48, and 72 h after transfection. FCM results showed that the VEGF-siRNA group had an apparent aneuploid peak.

Conclusion: VEGF, FLT-1 and FLK-1 are associated with colorectal carcinogenesis. siRNA silencing of the VEGF gene suppresses proliferation, and induces apoptosis in HCT116 cells. The results suggest that VEGF may be a new gene therapy target for colorectal cancer.