• CRISPR/Cas9
• RNAi strategies
• antisense oligonucleotide (ASO)
• aptamer
• riboswitch
• ribozyme
• small interfering RNA (siRNA)

• Carcinoma, Hepatocellular/therapy
• Cell-Free System
• Cloning, Molecular
• Genetic Therapy/methods
• Genetic Vectors
• Humans
• In Vitro Techniques
• Liver Neoplasms/therapy
• Nucleic Acid Conformation
• Point Mutation
• RNA, Catalytic/chemistry
• RNA, Catalytic/genetics
• RNA, Catalytic/metabolism
• RNA, Messenger/metabolism
• Tumor Suppressor Protein p53/genetics

AIM: To investigate the effect of antisense RNA to vascular endothelial growth factor165 (VEGF₁₆₅) on human esophageal squamous cell carcinoma cell line EC109 and the feasibility of gene therapy for esophageal carcinoma.

METHODS: By using subclone technique, the full length of VEGF₁₆₅ amino acid cDNA, which was cut from pGEM-3Zf(+), was cloned inversely into the eukaryotic expression vector pCEP4.The recombinant plasmid pCEP-AVEGF₁₆₅ was transfected into EC109 cell with lipofectamine. After a stable transfection, dot blot, enzyme-linked immunosorbent assay (ELISA), laser confocal imaging system analysis, transmission electron microscopy and flow cytometry were performed to determine the biological characteristics of EC109 cell line before and after transfection in vitro and whether there was a reversion in the tumorigenic properties of the EC109 cell in vivo.

RESULTS: The eukaryotic expression vector pCEP-AVEGF₁₆₅ was successfully constructed and transfected into EC109 cells. The expression of VEGF₁₆₅ was significantly decreased in the transfected cells while the biological characteristics of the cells were not influenced by the expression of antisense gene. The tumorigenic and angiogenic capabilities were greatly reduced in nude mice, as demonstrated by reduced tumor end volume (820 ± 112.5) mm³vs (7930 ± 1035) mm³ and (7850 ± 950) mm³,P£¼0.01£½ and microvessel density(8.5 ± 1.2) mm^-2vs (44.3 ± 9.4) mm^-2 and (46.4 ± 12.6) mm^-2,P < 0.01) in comparison between experimental groups empty vector transfected group and control group.

CONCLUSION: The angiogenesis and tumorigenicity of human esophageal squamous cell carcinoma were effectively inhibited by VEGF₁₆₅ antisense RNA. Antisense RNA to VEGF₁₆₅ can potentially be used as an adjuvant therapy for solid tumors.

• Animals
• Carcinogenicity Tests
• Carcinoma, Squamous Cell/physiopathology
• Carcinoma, Squamous Cell/therapy
• Cell Cycle/genetics
• Cloning, Molecular
• Endothelial Growth Factors/genetics
• Esophageal Neoplasms/physiopathology
• Esophageal Neoplasms/therapy
• Gene Expression Regulation, Neoplastic
• Genetic Therapy
• Humans
• Lymphokines/genetics
• Mice
• Mice, Inbred BALB C
• Mice, Nude
• Microscopy, Electron
• Neovascularization, Pathologic/prevention & control
• Neovascularization, Pathologic/therapy
• Plasmids
• RNA, Antisense/pharmacology
• Transfection
• Tumor Cells, Cultured/physiology
• Tumor Cells, Cultured/ultrastructure
• Vascular Endothelial Growth Factor A
• Vascular Endothelial Growth Factors