Research Paper Volume 12, Issue 5 pp 4247—4267

Downregulation of lung miR-203a-3p expression by high-altitude hypoxia enhances VEGF/Notch signaling

Figure 8. VEGF-A expression is rescued by miR-203a-3p knockdown. (A) The validation of VEGF-A expression reduction in VEGF-shRNA-transduced cells by qRT-PCR. (B) Western blotting data showing reduced expression of VEGF-A in PMVECs transduced with VEGF-shRNA. (C) Morphological evaluation in PMVECs transduced with VEGF-shRNA+ASO-NC (negative VEGF-shRNA+ASO control) or VEGF-shRNA+ASO (miR-203a-3p knockdown). Note that inhibition of miR-203a-3p activity reversed morphological impairment caused by VEGF silencing. (D) Flow cytometry apoptosis assay indicates increased apoptosis in PMVECs expressing VEGF-shRNA, and reversal of the effect by ASO-mediated miR-203a-3p silencing. (EF) CCK-8 assay showing improved viability in PMVECs expressing VEGF-shRNA+ASO, compared with the VEGF-shRNA+ASO-NC group. (G) qRT-PCR results showing reduced expression of VEGF-A and its downstream mediators, VEGFR2 and Hes-1, in cells transfected with VEGF-shRNA. The expression of these mRNAs increased instead in cells co-expressing miR-203a-3p-ASO. (H) Western blotting results showing protein expression changes consistent with the mRNA data showed in (G). Data are mean ± SEM. *P < 0.05, **P < 0.01 compared with VEGF-shRNA negative control (VEGF-shRNA-NC) and normoxia groups; #P < 0.05, ##P < 0.01 compared with the VEGF-shRNA or 24-h hypoxia group; &P < 0.05, &&P < 0.01 compared with the VEGF-shRNA+ASO-NC or 48-h hypoxia group.