Figure 3
Experimental confirmation of β1AR, β2AR, BNIP3L and Giα-2 as miR-30 targets. (a) RT-qPCR shows relative target expression levels when overexpressing miR-30. All values are normalized to U6 levels and presented as ratios to the pre-NC-transfected controls (20 nmol/l). Three biological replicates were performed on H9c2 cultures. (b) Relative protein levels of BNIP3L and Giα-2 were measured by western blot; band densitometry was quantified by ImageJ and normalized to tubulin. (c) Schematic view of the sponge vector designed for miR-30 family inhibition (pEGFP-sp30), using the pEGFP-C1 plasmid as backbone. EGFP expression in transfected cultures was detected by microscopy. (d) Relative target mRNA levels measured by RT-qPCR and protein levels (e) upon transfection with pEGFP-sp30 for miR-30 family inhibition, or the control vector pEGFP-C1. Data are averages of three biological replicates performed on H9c2 cardiac cells. (f) Target expression levels measured by RT-qPCR following DOX treatment (1 μmol/l, 18 h), alone or in combination with miR-30e overexpression (pre-30e transfection). Three independent replicates were performed on H9c2 cells, being presented as averaged ratio to control ±S.E.M. (g) RT-qPCR data showing expression levels of the predicted miR-30 targets in vivo in DOX-treated hearts (15 mg/kg cumulative dose, n=5 rats per group). Student’s t-test performed for statistical analysis. Error bars represent±S.E.M.
