Research Article

Inhibition of vascular endothelial growth factor A expression in mouse granulosa cells by lentivector-mediated RNAi

Published: November 28, 2012
Genet. Mol. Res. 11 (4) : 4019-4033 DOI: 10.4238/2012.November.28.1

Abstract

Vascular endothelial growth factor (VEGF) has been found responsible for the induction of proliferation and differentiation in granulosa cells. We constructed four short hairpin RNA (shRNA) expression plasmids targeting the mouse VEGFA gene, and examined their effect on VEGF expression in mouse granulosa cells (MGC) in vitro. Four different shRNA oligonucleotides targeting the coding sequence of mouse VEGFA mRNA and one negative control (shNC) were designed and cloned into a pGPU6/GFP/Neo siRNA expression vector, and transiently transfected into MGC. At 48 h post-transfection, total RNA was extracted from the cells and subjected to qRT-PCR analysis. The most effective interference vector, shVEGF1487 was chosen for lentiviral construction. The recombinant plasmid was then transfected into 293FT cells via LipofectamineTM 2000-mediated gene transfer, for the production of lentivirus, and then concentrated via ultracentrifugation. This lentiviral vector was then used for the transduction of MGC. VEGFA gene expression, apoptosis genes and VEGFA receptor genes were detected by qRT-PCR, the VEGFA protein level in culture media by ELISA assay and protein levels in MGC by Western blot analysis. The four VEGFA expression plasmids were successfully constructed and the most effective interference vector, shVEGF1487, was chosen for lentiviral production and MGC transduction. There was significant knockdown of the VEGFA gene, receptor genes and apoptosis genes for all the shVEGF constructs, compared with the shNC and Mock controls. The lentiviral vector also gave significant knockdown of the VEGFA gene. Protein levels were lower for most of the shVEGFs based on Western blot analysis with exception of VEGF1359; in this case, it was higher than shNC but lower than for the Mock group. Lentivector-transduced MGC also gave lower levels of protein. We conclude that shVEGF expression plasmids and lentivector carrying RNAi are promising tools for the inhibition of VEGF, the corresponding receptor genes, and apoptosis gene expression in MGC.

Vascular endothelial growth factor (VEGF) has been found responsible for the induction of proliferation and differentiation in granulosa cells. We constructed four short hairpin RNA (shRNA) expression plasmids targeting the mouse VEGFA gene, and examined their effect on VEGF expression in mouse granulosa cells (MGC) in vitro. Four different shRNA oligonucleotides targeting the coding sequence of mouse VEGFA mRNA and one negative control (shNC) were designed and cloned into a pGPU6/GFP/Neo siRNA expression vector, and transiently transfected into MGC. At 48 h post-transfection, total RNA was extracted from the cells and subjected to qRT-PCR analysis. The most effective interference vector, shVEGF1487 was chosen for lentiviral construction. The recombinant plasmid was then transfected into 293FT cells via LipofectamineTM 2000-mediated gene transfer, for the production of lentivirus, and then concentrated via ultracentrifugation. This lentiviral vector was then used for the transduction of MGC. VEGFA gene expression, apoptosis genes and VEGFA receptor genes were detected by qRT-PCR, the VEGFA protein level in culture media by ELISA assay and protein levels in MGC by Western blot analysis. The four VEGFA expression plasmids were successfully constructed and the most effective interference vector, shVEGF1487, was chosen for lentiviral production and MGC transduction. There was significant knockdown of the VEGFA gene, receptor genes and apoptosis genes for all the shVEGF constructs, compared with the shNC and Mock controls. The lentiviral vector also gave significant knockdown of the VEGFA gene. Protein levels were lower for most of the shVEGFs based on Western blot analysis with exception of VEGF1359; in this case, it was higher than shNC but lower than for the Mock group. Lentivector-transduced MGC also gave lower levels of protein. We conclude that shVEGF expression plasmids and lentivector carrying RNAi are promising tools for the inhibition of VEGF, the corresponding receptor genes, and apoptosis gene expression in MGC.