Effects Of Vascular Endothelial Growth Factor In Leukemia/tumor Genesis And Development

Objectives: The purpose of this study was to explore the effects of VEGF gene on proliferation, apoptosis, and chemosensitivity of leukemic cells and tumor cells when VEGF was suppressed or overexpressed. We look for the highly efficient and specific VEGFsiRNAs, which can cause downregulation of VEGF gene expression, perhaps as medicines for gene therapy of leukemia and tumors.Methods: We gained the sequence of VEGFmRNA from GeneBank. The nine VEGF siRNAs were designed by using siRNA guidelines and RNAstructure software.The lipofectamine^TM2000 was used to transfect VEGFsiRNAs into cells. The inhibition effect of VEGFsiRNA alone or in combination with chemotherapy drugs were analysed by CCK8 method. The level of VEGFmRNA was detected by reverse transcription and quantitative polymerase chain reaction (RT-PCR). The level of VEGF protein was determined by ELISA and Western Blot. The cell cycle and cell apoptosis were detected by flow cytometry. The recombinant eukaryotic express plasmid pEGFP-C1-hVEGF₁₆₅ was constructed with conventional gene engineering methods. The pEGFP-C1-hVEGF₁₆₅ was transfected into leukemic k562 cells and it's biological effects were evaluated.Results: A quantitative parameter called H-b index can characterize the secondary structure of VEGFmRNA. There was negative correlation between the suppression of VEGF protein expression and the H-b index(r=-0.498). The siRNAs showed a strong RNAi activity with A residue at position 6 and A/U residues at position 19 of sense strand of siRNAs. We have screened two optimal siRNA sequences, named as VEGFsiRNA2 and VEGFsiRNA7, which can inhibit the growth and proliferation of liver cancer cell SMMC7721,leukemic k562 cell and h160 cell, downregulate the expression of VEGFmRNA and VEGF protein. The inhibition effects of siRNAs were better than that of antisense oligodeoxynucleotide. VEGFsiRNA can't induce the cells to apoptosis. VEGFsiRNA can increase the sensitivity of leukemic cells k562,hi60 and Molt4 to Ara-c and HHT. VEGFsiRNA combined with Ara-c and HHT can effectively reduced the level of VEGF protein of leukemic cells. Compared with the k562 cells transfected pEGFP-C1, the k562 cells transfected pEGFP-Cl-hVEGF₁₆₅ grew faster and expression of VEGFmRNA and VEGF protein increased significantly. In addition, k562 cells transfected pEGFP-C1-hVEGF₁₆₅ had relatively higher cell survival rate to chemotherapy drugs.Conclusions: (1) VEGFsiRNAs can reduce the level of VEGFmRNA expression and VEGF protein, and inhibit the proliferation of tumor cells and leukemic cells. (2) VEGFsiRNA can increase the sensitivity of leukemic cells to Ara-c and HHT. (3) The overexpression of VEGF can promote the proliferation of leukemic cells and decrease the sensitivity of leukemic cells to Ara-c, HHT and FU. There is an important effect of vascular endothelial growth factor on proliferation and drug-sensitivity of leukemia.