To explore the mechanism by which m

To explore the mechanism by which miR-134-5p promotes HG-induced apoptosis of podo-cytes, we searched two miRNA target analyzingdatabases (TargetScan and miRNAWalk 2.0)and found that bcl-2 is a latent target of miR-134-5p. We then performed luciferase reporterassays. The sequence of the 3’-UTR of bcl-2mRNA matched the seed sequence of miR-134-5p. To test the functional significance ofthis finding, the 3’-UTR sequences, containingputative binding sites of the WT or mut for theseed matching sites, were introduced into aluciferase reporter vector (Figure 3A) and eachwas co-transfected into podocytes with themiR-134-5p mimic or miR-ctrl (Figure 3B). Theresults demonstrated that bcl-2 is a direct tar-get of miR-134-5p. To further examine theeffect of miR-134-5p on bcl-2, we transfectedHG-treated podocytes with anti-miR-134-5p oranti-miR-ctrl. Western blotting was performedto assess bcl-2 protein levels. The resultsshowed that bcl-2 protein levels were increased in HG-treated podocytes transfected with anti-miR-134-5p compared with that in cells trans-fected with anti-miR-ctrl (Figure 3C, 3D).Overexpression of miR-134-5p by miR-134-5pmimic transfection in podocytes reduced theexpression of bcl-2 under NG conditions (FigureS2). Together, these results indicate that bcl-2is a direct target of miR-134-5p.