Abstract:

Objective To explore the effects of miR-20b on the expression of vascular endothelial growth factor (VEGF) in RAW264.7 cells of rats. Methods RAW264.7 cells of mice were stimulated by different concentrations (1, 10, 50, 100, and 200 ng/mL) of TNF-α for 24 h and the amount of VEGF in the supernatant was detected by the ELISA. The miR-20b and VEGF mRNA expressions were detected by the Real-Time PCR for RAW264.7 cells stimulated by TNF-α (50 ng/mL) for 6 h. The Pictar algorithm was used to predict whether VEGF was a regulating target of miR-20b. MiR-20b mimics and its control were transfected into RAW264.7 cells by Lipofectamine 2000. ELISA was used to detect the amount of VEGF in the supernatant after the transfected RAW264.7 cells were stimulated by TNF-α (100 ng/mL) for 24 h. Results TNF-α induced the RAW264.7 cells to secret VEGF and 50-100 ng/mL TNF-α was the most suitable concentration for stimulation. After RAW264.7 cells were stimulated by TNF-α (50 ng/mL) for 6 h, the expression of VEGF was elevated to (1.60±0.85) times of that of the control group, while the expression of miR-20b was decreased to (0.55±0.33) times of that of the control group. The Pictar algorithm showed that the VEGF 3′-UTR contained the complementary sequences ‘GCACUUU’ of miR-20b seed region ‘AAAGUGC’. The increased expression of VEGF protein was completely inhibited after TNF-α-stimulated RAW264.7 cells were transfected with miR-20b mimics (P＜0.01), while for the control group, transfected with miR-20b mimics had no significant effect on the production of VEGF (P>0.05). Conclusion MiR-20b can negatively regulate the expression of VEGF in RAW264.7 cells of mice.

Key words: miR-20b, vascular endothelial growth factor, RAW264.7 cells