Overexpression of HER2 or HER3 in ovarian cancer cells reversed miR-199a and miR-125b inhibitory effects on Akt/p70S6K1/HIF-1α/VEGF pathway.

<p>(A, B) A2780 vector, A2780 HER2, OVCAR-3 vector and OVCAR-3 HER3 cells were cultured under normoxic condition, and were transfected with pre-miR control, pre-miR-199a and pre-miR-125b precursor at the final concentration of 30 nM. The cells were harvested and analyzed 70 h after transfection for protein levels of p-Akt, total Akt, p-p70S6K1, p70S6K1, HIF-1α and HIF-1β. (C) RT-PCR was used to assess the VEGF mRNA levels in stable cells transfected with miR-control, miR-199a or miR-125b. (D) Cells were seeded in 12-well plates and transiently co-transfected with human VEGF luciferase reporter, pre-miRNAs and β-galactosidase plasmids. The cells were cultured for 48 h after transfection and relative luciferase activities were measured as described in Method section. (E) A2780 cells stably overexpressing HIF-1α was established by infecting A2780 cells with retrovirus carrying HIF-1α. (F) A2780 cells and A2780/HIF-1α cells were transiently transfected with the indicated miRNAs, and harvested 70 h later to analyze VEGF mRNA levels by RT-PCR as indicated in the figure. (G) A2780 vector control cells and A2780 HIF-1α cells were transfected with pre-miR-control, pre-miR-199a or pre-miR-125b, respectively; then 2×10⁶ cells were used to perform angiogenesis assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056647#pone-0056647-g002" target="_blank">Figure 2</a>. *Significantly different compared with that of the vector cell line transfected with pre-miR-control (P<0.05).