Figure 4. miR-146a and miR-150 enhance spheroid formation.

24 hours after transfection with anti-miR LNA inhibitors or pre-miR mimics as indicated, 700/µl SKOV-3 or 600/µl OVCAR-8 cells were seeded in 35 well agarose micromolds with one spheroid forming in each well. A. miR-21 and miR-146a are up-regulated in 4 day SKOV-3 and OVCAR-8 spheroids detected using Taqman qPCR array cards in two replicates. Error bars are s.e.m. B. Inhibition of miR-146a with 10 nM LNA delays spheroid formation and leads to more amorphous and looser formed spheroids in SKOV-3 and OVCAR-8 after 2 days. Red bar is 400 µm. C. Box and whisker plot shows that inhibition of miR-146a with LNAs significantly reduces spheroid size after 4 days in SKOV-3, and modestly in OVCAR-8. Representative expression shown from four replicates. D. Ectopic expression of miR-150 and miR-146a significantly enhances spheroid formation after 4 days. SKOV-3 and OVCAR-8 cells were transfected with 50 nM of pre-miR miR-150 and miR-146a pre-miRs before spheroid formation. SKOV-3 or OVCAR-8 cells were transfected as indicated. Representative spheroids are shown. Red bar is 400 µm. Box and whisker plots of the size distribution of 45 spheroids from a representative experiment. Experiment was reproduced three times. P-values determined by Student's t-test.