Inhibition Of Vascular Endothelial Growth Factor A (VEGFA)Expression In MGC By Lentivector-mediated RNAi

Vascular Endothelial Growth Factor (VEGF) is a sub-family of the platelet-derived growth factor (PDGF) of cystine-knot growth factors, which function as a signal protein produced by cells to stimulate vasculogenesis and angiogenesis. VEGF’s normal function is to create new blood vessels during embryonic development, new blood vessels after injury, muscle following exercise, and new vessels to bypass blocked vessels. It has been found that VEGF is responsible for the induction of proliferation and differentiation functions on granulosa cells and its up-regulation (knockin) or down-regulation (knockdown) by RNA interference (RNAi) provides a powerful tool to study its function in details in these cells. RNAi is a sequence-specific posttranscriptional gene silencing mechanism, initiated by the introduction of double-stranded RNA (dsRNA) of a specific gene of interest. Recently, researchers had used short hairpin RNA (shRNA) into lentiviral vectors to deliver siRNA successfully into mammalian cells to obtain stable gene silencing. VEGF shRNA can be used as potential strategy to knockdown VEGF gene, and check its effects on its receptor and apoptosis genes in mouse granulosa cells (MGC).In this experiment we construct four short hairpin RNA (shRNA) expression plasmid targeting mouse VEGFA gene, and explore its effects on VEGF expression on MGC in vitro. Four different shRNA oligonucleotides targeting the coding sequence of mouse VEGFA mRNA and one negative control were designed and cloned into pGPU6/GFP/Neo siRNA expression vector, and transiently transfected into MGC. At48hours post-transfection, total RNA was extracted from cells and subjected to qRT-PCR analysis. The most effective interference vector, shVEGF1487was chosen for lentiviral construction. The recombinant plasmid was then transfected into293FT cells via lipofectamineTM2000mediated 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, VEGFA protein level in culture media by ELISA assay and protein level in MGC by Western blot analysis. The four VEGFA expression plasmids were successfully constructed and most effective interference vector, shVEGF1487, was chosen for lentiviral production and MGC transduction. There were significant knockdown of the VEGFA gene, receptor genes and apoptosis genes were reduced for all the shVEGFs constructs, compared with the shNC and Mock controls. The lentiviral vector also shows significant knockdown of the VEGFA gene. Protein concentration in media was lower than shNC and Mock group against all the shVEGFs. Protein level was lowered for most of the shVEGFs during Western blot analysis with exception of VEGF1359, was higher than the shNC but lower than the Mock. Lentivector transduced MGC also showed low level of protein. These findings suggest that shVEGF expression plasmids, and lentivector, mediated RNAi is a promising tool for the inhibition of the VEGF, its receptor genes and apoptosis genes expression in MGC.