Fig. 6: MiR-133a directly targets multiple components of TGF-β1 signaling pathways.

a KEGG analysis of Targetscan predicted miR-133a targets. HFL cells were transfected with miR-133a mimic or control mimic (CTL) for 24 h, and then stimulated without or with 1 ng/mL TGF-β1 for 48 h. Cells were harvested and subjected to western blot (b–d) or quantitative RT-PCR analysis (e, f). Transfection of miR-133a down-regulated TGFBR1 protein expression (b), blocked TGF-β1-stimulated Smad2/3 phosphorylation (c), CTGF protein expression (d), Col1a1 (e), and Col4a1 (f) mRNA expression. Data are mean ± SEM (n = 3–4) with *P < 0.05; ***P < 0.001. g The 3′UTR of TGFBR1, CTGF or Col1a1 contains a putative miR-133a binding site that is conserved among different species (in red). miR-133a binding site mutations were created by deletion or nucleotide changes (in blue). h The 3′UTR fragments containing the putative miR-133a binding site (3’UTR-133a) or their mutants (3′UTR-133aM) of human TGFBR1 (inserted fragment: 1989–2403; seeding region: 2161–2167), CTGF (inserted fragment: 886–1065; seeding region: 1027–1033), or Col1a1 (inserted fragment 78–301; seeding region: 194–200) were subcloned into the pmirGLO reporter vector. i These luciferase reporter constructs were transfected into HEK293 cells, along with control or miR-133a mimic. Cells were harvested for firefly luciferase (fLuc) assays with Renilla luciferase (rLuc) as an internal control. Data are mean ± SEM (n = 3–4) with **P < 0.01