FIG. 3.

miR-155 mediates the effect of TGF-β on EMT. (A) Ectopic expression and knockdown of miR-155. NMuMG cells were transfected with the indicated plasmids and oligonucleotides. Following treatment with (+) or without (−) TGF-β, cells were subjected to Northern blot analysis with [α-³²P]dATP-labeled miR-155 and U6 probes. Pre miR-155, pcDNA6.2-GW/miR-155; Pre miR-ctrl, pcDNA6.2-GW/miR-control. (B and C) The knockdown of miR-155 inhibited TGF-β-induced EMT and tight junction dissolution. NMuMG cells were transfected with miR-155 ASO and control ASO and then treated with or without TGFβ for 24 h. Cell morphologies were documented using a phase-contrast microscope (B), and cells were stained with anti-ZO-1 and anti-E-cadherin antibodies conjugated to fluorescein isothiocyanate and tetramethyl rhodamine isothiocyanate, respectively (C). Arrows in panel C indicate the restoration of TGF-β-disrupted tight junctions by the knockdown of miR-155. (D) The knockdown of miR-155 inhibits the TGF-β downregulation of E-cadherin (E-Cad). NMuMG cells were transfected with control and miR-155 ASO, treated with TGF-β for 24 h, and then immunoblotted with the indicated antibodies. (E and F) The overexpression of miR-155 disrupted proper tight junction formations and accelerated TGF-β-induced EMT. Stably miR-155-transfected and control NMuMG cells were treated with or without TGF-β for 12 h and photographed (E) and immunofluorescence-stained with the indicated antibodies (F). The tight junction dissolution promoted by the ectopic expression of miR-155 is indicated by arrows. (G) Western blot analysis of E-cadherin in NMuMG cells that were transfected with miR-155 and then treated with TGF-β for 12 h.