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. 2016 Apr 28;6:24900. doi: 10.1038/srep24900

Figure 5. Identification of RhoB and MLC2 as direct targets of miR-223.

Figure 5

Luciferase reporter gene assay on the interaction between miR-223 and 3′-UTR of RhoB (A) and MLC2 (B) in HEK293 cells. Upper panel: putative miR-223 binding sites in the 3′-UTR of RhoB or MLC2 along with the mutation sites. TS1,target site 1 (a pooly conserved binding site); TS2, target site 2 (a conserved binding site); Bottom panel: 3′-UTR luciferase reporter assay with targets and their mutant along with miR-223/miR-Con overexpressing vectors. Bar charts of luciferase reporter analysis represent means ± SE (n = 3), firefly luciferase activity was normalized to renilla luciferase activity. (C) Active RhoB proteins in miR-223 or miRNA control overexpressing PASMC were pulled down by rhotekin-RBD beads and detected by western blotting analysis using anti-RhoB antibody (n = 3). (D) Expression of RhoB/Rho-kinase signaling pathway related components in PASMC, with overexpression or inhibition of miR-223, was measured by western blotting (n = 3). (E) Expression of RhoB, MLC2 and PCNA in lungs of hypoxia-exposed rats administered agomirs miR-223/miR-Con was determined by western blotting (n = 3). β-actin was used as a loading control for the above western blot analysis. Two representative blots are shown. *p < 0.05, **p < 0.01.