Research Paper Volume 14, Issue 15 pp 6255—6268

Figure 4. cMAP4K2 regulates endothelial cell function by acting as the miRNA sponge. (A) The cell fractions were isolated from HRVECs and immunoprecipitated using Ago2 or IgG antibody. The amount of cMAP4K2 in the immunoprecipitate was examined by qRT-PCRs (n = 4, ^*P < 0.05). (B) HRVEC were transfected with pGL3-Basic (Ctrl) or LUC-cMAP4K2 with different miRNA mimic and pRL-TK vector (internal transfection control). Luciferase activity was detected at 36 h post-transfection using the Dual-Luciferase Reporter Assay kit (n = 4, ^*P < 0.05). (C) The schematic figure showed the potential binding regions of miR-377 on cMAP4K2 transcript. (D) The biotinylated miR-21 or miR-377 were transfected into HRVECs. The amount of cMAP4K2 and cSPECC1 (negative control) in the input and bound fractions were examined by qRT-PCR assays following streptavidin capture. The relative immunoprecipitate (IP)/input ratios were plotted (n = 4). (E) LUC-VEGFA or LUC-VEGFA-mutant was co-transfected without or with miRNA mimic and pRL-TK vector. Luciferase activity was detected at 36 h after transfection (n = 4). MUT: mutated VEGFA 3’-UTR without miR-377 binding site. (F) HRVECs were cultured with D-glucose (25 mM) for 24 h and then transfected with scramble (Scr) mimic, miR-377 mimic, Scr siRNA, cMAP4K2 siNRA, or left untreated (Ctrl) for 24 h. qRT-PCRs and western blots were performed to detect the expression of VEGFA at mRNA levels and protein levels (n = 3). (G) Diabetic retinas received an injection of Src agomir, miR-377 agomir, Scr shRNA, cMAP4K2 shNRA, or left untreated (Ctrl) for 1-month. qRT-PCRs and western blots were performed to detect the expression of VEGFA at mRNA levels and protein levels (n = 3 animals per group). ^*P < 0.05. The significant difference was analyzed by Student t test (A), one-way (B, F, and G), or two-way ANOVA (D and E) followed by the Bonferroni post hoc test.