American Society of Hematology

Figure 5

$Figure 5. miR-130a antagonizes the antiangiogenic activity of GAX. (A) miR-130a antagonizes G0/G1 cell-cycle arrest attributable to GAX depending on the presence of its consensus sequences in the GAX 3′-UTR. HUVECs rendered quiescent by serum starvation overnight were transfected with empty vector or pcDNA-3.1-miR-130a and either pcDNA3.1-GAX or pcDNA3.1-GAX-3′-UTR as for the tube formation experiment and exposed to 10% FBS for 24 hours, after which cells were harvested, stained with PI, and subjected to flow cytometry to determine cell-cycle distribution. By definition, the change in the G0/G1 fraction for the empty vector control is 0, and the G0/G1 fraction for the vector control was 0.38 in the experiment displayed. Statistical significance was determined by one-way ANOVA, and both the miR-130a and GAX-3′-UTR + miR-130a groups showed a statistically significant (P < .01) change from the others. (B) miR-130a antagonizes the antimigration activity of GAX. HUVECs were cotransfected with either pcDNA3.1-GAX-3′-UTR or pcDNA3.1-GAX plus either pcDNA3.1-miR130a or pcDNA3.1 control empty vector and plated 18 hours later onto 8.0 μm pore size polycarbonate membranes in 24-well plates and allowed to attach, after which migration assays were carried out as described in “Migration and tube formation assays.” Cells were counted in 5 hpf per well (*P < .01 compared with empty vector control). (C) miR-130a antagonizes the antiangiogenic activity of GAX. HUVECs were cotransfected with either empty vector or pcDNA3.1-miR-130a and pcDNA3.1-GAX-3′-UTR or pcDNA3.1-GAX and then incubated overnight, after which tube formation assays were carried out. The ratio of cotransfection was 2:1 miR-130a:GAX expression construct. Tube counts were determined as described in “Migration and tube formation assays.” (D) Quantification of tube number per low-powered field (tubes/LPF; *P < .01). (E and F) An inhibitor of miR-130a reverses its antagonism of GAX activity. Experiments described in panels C and D were repeated, but with the addition of 100 nM miR-130a inhibitor. * indicates P < .01. Error bars represent SEM.$

miR-130a antagonizes the antiangiogenic activity of GAX. (A) miR-130a antagonizes G₀/G₁ cell-cycle arrest attributable to GAX depending on the presence of its consensus sequences in the GAX 3′-UTR. HUVECs rendered quiescent by serum starvation overnight were transfected with empty vector or pcDNA-3.1-miR-130a and either pcDNA3.1-GAX or pcDNA3.1-GAX-3′-UTR as for the tube formation experiment and exposed to 10% FBS for 24 hours, after which cells were harvested, stained with PI, and subjected to flow cytometry to determine cell-cycle distribution. By definition, the change in the G₀/G₁ fraction for the empty vector control is 0, and the G₀/G₁ fraction for the vector control was 0.38 in the experiment displayed. Statistical significance was determined by one-way ANOVA, and both the miR-130a and GAX-3′-UTR + miR-130a groups showed a statistically significant (P < .01) change from the others. (B) miR-130a antagonizes the antimigration activity of GAX. HUVECs were cotransfected with either pcDNA3.1-GAX-3′-UTR or pcDNA3.1-GAX plus either pcDNA3.1-miR130a or pcDNA3.1 control empty vector and plated 18 hours later onto 8.0 μm pore size polycarbonate membranes in 24-well plates and allowed to attach, after which migration assays were carried out as described in “Migration and tube formation assays.” Cells were counted in 5 hpf per well (*P < .01 compared with empty vector control). (C) miR-130a antagonizes the antiangiogenic activity of GAX. HUVECs were cotransfected with either empty vector or pcDNA3.1-miR-130a and pcDNA3.1-GAX-3′-UTR or pcDNA3.1-GAX and then incubated overnight, after which tube formation assays were carried out. The ratio of cotransfection was 2:1 miR-130a:GAX expression construct. Tube counts were determined as described in “Migration and tube formation assays.” (D) Quantification of tube number per low-powered field (tubes/LPF; *P < .01). (E and F) An inhibitor of miR-130a reverses its antagonism of GAX activity. Experiments described in panels C and D were repeated, but with the addition of 100 nM miR-130a inhibitor. * indicates P < .01. Error bars represent SEM.

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