Fig. 4. miRNAs selectively regulate survivin isoform expression.

A: Selected, predicted miRNA-binding sites in survivin isoform 3′ UTRs. B: qRT-PCR for selected microRNAs in H9 cells shows that miR-203, -218, -542-5p, and -335 are more highly expressed in h9 cells grown in differentiation medium compared to in pluripotency medium. miR-338 expression was equivalent in the two media and miR-135a expression was lower in differentiation vs. pluripotency medium. C: Western blot showing the effects of miRNA inhibitors on survivin isoforms and the pluripotency marker Nanog in cells grown in differentiation medium. Cells transfected with the miR-203 inhibitor displayed increased levels of nuclear, but not cytoplasmic survivin, relative to control-transfected cells; Nanog levels were also higher. Inhibition of miR-135a resulted in decreased levels of both nuclear and cytoplasmic survivin, as well as Nanog. D: Western blot showing that cells grown in pluripotency medium and transfected with miR-203 precursor, to mimic miR-203 overexpression, showed decreased levels of nuclear survivin and Nanog, but no change in cytoplasmic survivin levels. E: Normalized luciferase signal in cells transiently co-transfected with short- and medium-length 3′ UTR luciferase constructs and miR-203 inhibitor. Show that only the medium-length 3′ UTR from the ΔEx3/nuclear survivin isoform is affected by mir-302 inhibitor.