miR-130a down-regulates GAX expression in HUVECs. (A) miR-130a down-regulates GAX mRNA expression. HUVECs were transfected with either empty vector (pcDNA3.1) or miR-130a expression vector (pcDNA3.1-miR-130a) and then incubated overnight in LSM ( = 0.1% FBS), after which total RNA was isolated for QRT-PCR. miR-130a expression markedly down-regulated the expression of endogenous GAX. (B) Representative Western blot of protein from the same experiment as in A. Band intensity was measured by densitometry and normalized to α-tubulin levels. Endogenous GAX protein is also down-regulated by miR-130a. * indicate P < .01. (C,D) Inhibition of GAX expression attributable to miR-130a depends on the presence of the miR-130a binding sites. HUVECs were cotransfected with either pcDNA3.1 or pcDNA3.1-miR-130a plus either pcDNA3.1 or pcDNA3.1-GAX-3′-UTR. Exogenous GAX protein from these plasmids was detected using anti-Flag antibody as described in “miR-130a binding sites in the GAX 3′-UTR mediate serum-induced down-regulation.” Down-regulation of GAX expression by miR-130a was only observed with the construct containing the 2 miR-130a consensus sequences in the 3′-UTR. (C = QRT-PCR; D = Western blot.) (E) Inhibition of miR-130a blocks the down-regulation of GAX attributable to mitogens. HUVECs were transfected with 0 to 100 nM mir-130a inhibitor and incubated in fresh culture medium overnight, after which they were placed in 0.1% FBS or 2% RBS medium for 6 hours. Total RNA was isolated for QRT-PCR as described in insert section. Error bars represent SEM.