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Blocking effects of siRNA on VEGF expression in human colorectal cancer cells
Yu Yin1, Li-Yu Cao, Wen-Qing Wu
1Department of Pathology, Anhui Medical University, Hefei 230032, Anhui Province, China.
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.
Insights
Vascular Endothelial Cell Growth Factor (VEGF) and its receptors are elevated in colorectal cancer. Small interfering RNAs (siRNAs) targeting VEGF effectively suppressed tumor cell proliferation and induced apoptosis, suggesting VEGF as a gene therapy target.
Area of Science:
- Oncology
- Molecular Biology
- Gene Therapy
Background:
- Colorectal carcinoma (CRC) is a significant health concern.
- Vascular Endothelial Cell Growth Factor (VEGF) and its receptors, Fms-like tyrosine kinase 1 (FLT-1) and fetal liver kinase 1 (FLK-1), play crucial roles in tumor angiogenesis and progression.
Purpose of the Study:
- To investigate the expression levels of VEGF, FLT-1, and FLK-1 in colorectal carcinoma tissues compared to normal tissues.
- To evaluate the efficacy of small interfering RNAs (siRNAs) in silencing VEGF expression and its impact on colorectal cancer cell behavior.
Main Methods:
- Immunohistochemical staining was used to assess protein expression in 82 CRC cases and 14 normal mucosae.
- VEGF-targeting siRNAs were synthesized and transfected into HCT116 colorectal cancer cells.
- Cell proliferation was measured using MTT assays, and apoptosis was analyzed by flow cytometry (FCM).
Main Results:
- VEGF, FLT-1, and FLK-1 expression were significantly higher in CRC tissues than in normal tissues (P < 0.001).
- VEGF expression correlated positively with lymph node metastasis and clinical stage (P < 0.05).
- VEGF-siRNA transfection significantly reduced VEGF protein levels, inhibited HCT116 cell proliferation, and induced apoptosis.
Conclusions:
- VEGF, FLT-1, and FLK-1 are implicated in colorectal carcinogenesis.
- siRNA-mediated silencing of VEGF suppresses proliferation and induces apoptosis in colorectal cancer cells.
- VEGF represents a promising molecular target for novel gene therapies in colorectal cancer treatment.
