Original Article
Copyright ©2010 Baishideng. All rights reserved
World J Gastroenterol. Mar 7, 2010; 16(9): 1086-1092
Published online Mar 7, 2010. doi: 10.3748/wjg.v16.i9.1086
Blocking effects of siRNA on VEGF expression in human colorectal cancer cells
Yu Yin, Li-Yu Cao, Wen-Qing Wu, Hao Li, Yan Jiang, Hong-Fu Zhang
Yu Yin, Li-Yu Cao, Wen-Qing Wu, Hao Li, Yan Jiang, Hong-Fu Zhang, Department of Pathology, Anhui Medical University, Hefei 230032, Anhui Province, China
Wen-Qing Wu, Department of Pathology, Anhui Provincial Hospital, Hefei 230001, Anhui Province, China
Author contributions: Yin Y and Wu WQ performed the majority of the experiments; Yin Y and Cao LY wrote the manuscript; Li H, Jiang Y and Zhang HF revised the manuscript; Cao LY provided financial support for this work.
Supported by Natural Science Foundation of Anhui Province, No. 090413098
Correspondence to: Li-Yu Cao, Professor, Department of Pathology, Anhui Medical University, Hefei 230032, Anhui Province, China. caoliyu@yahoo.com.cn
Telephone: +86-551-5161130 Fax: +86-551-2841526
Received: September 15, 2009
Revised: December 17, 2009
Accepted: December 24, 2009
Published online: March 7, 2010
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.

Keywords: Colorectal carcinoma; Vascular endothelial cell growth factor; Fms-like tyrosine kinase 1; Fetal liver kinase 1; Small interfering RNA