OBJECTIVE: To investigate the effects of microRNA-145 (miR-145) on epithelial-mesenchymal transition (EMT) of TGF-β1-induced human renal proximal tubular epithelial (HK-2) cells. METHODS: The gene sequence of miR-145 was synthesized and cloned into pCMV-myc to construct recombinant plasmid pCMV-miR-145. HK-2 cells were divided into four groups: control (untreated), TGF-β1 (treated with TGF-β1), blank+TGF-β1 (treated with TGF-β1 after HK-2 cells transfected with blank plasmid) and miR-145+TGF-β1 (treated with TGF-β1 after HK-2 cells transfected with pCMV-miR-145 recombinant plasmid). Expression of miR-145 was detected by real-time PCR (RT-PCR). TGF-β1, Smad3, Smad2/3, p-Smad2/3, α-SMA, FN and type I collagen (Col I) protein levels were detected by Western blot. Concentrations of fibronectin (FN) and Col I in cell culture supernatants were measured using ELISA. RESULTS: pCMV-miR-145 recombinant plasmid was successfully transfected into HK-2 cells. Compared with the control group, the miR-145+TGF-β1 group showed a significant up-regulation in the expression level of miR-145 (P<0.01). However, the TGF-β1 and blank+TGF-β1 groups showed a significant down-regulation in the expression level of miR-145 compared with that in the control and miR-145+TGF-β1 groups (P<0.01). Compared with the TGF-β1 and blank+TGF-β1 groups, the miR-145+TGF-β1 group showed significantly reduced levels of the signal proteins TGF-β1, Smad3, Smad2/3 and p-Smad2/3 (P<0.05), as well as significantly reduced levels of the biomarkers α-SMA, FN and Col I (P<0.05). Meanwhile, concentrations of FN and Col I in cell culture supernatants also decreased (P<0.05). CONCLUSIONS: miR-145 modulates the EMT of HK-2 cells treated with TGF-β1, possibly by inhibition of the activation of TGF-β-dependent Smad signaling pathway.
OBJECTIVE: To investigate the effects of microRNA-145 (miR-145) on epithelial-mesenchymal transition (EMT) of TGF-β1-induced human renal proximal tubular epithelial (HK-2) cells. METHODS: The gene sequence of miR-145 was synthesized and cloned into pCMV-myc to construct recombinant plasmid pCMV-miR-145. HK-2 cells were divided into four groups: control (untreated), TGF-β1 (treated with TGF-β1), blank+TGF-β1 (treated with TGF-β1 after HK-2 cells transfected with blank plasmid) and miR-145+TGF-β1 (treated with TGF-β1 after HK-2 cells transfected with pCMV-miR-145 recombinant plasmid). Expression of miR-145 was detected by real-time PCR (RT-PCR). TGF-β1, Smad3, Smad2/3, p-Smad2/3, α-SMA, FN and type I collagen (Col I) protein levels were detected by Western blot. Concentrations of fibronectin (FN) and Col I in cell culture supernatants were measured using ELISA. RESULTS: pCMV-miR-145 recombinant plasmid was successfully transfected into HK-2 cells. Compared with the control group, the miR-145+TGF-β1 group showed a significant up-regulation in the expression level of miR-145 (P<0.01). However, the TGF-β1 and blank+TGF-β1 groups showed a significant down-regulation in the expression level of miR-145 compared with that in the control and miR-145+TGF-β1 groups (P<0.01). Compared with the TGF-β1 and blank+TGF-β1 groups, the miR-145+TGF-β1 group showed significantly reduced levels of the signal proteins TGF-β1, Smad3, Smad2/3 and p-Smad2/3 (P<0.05), as well as significantly reduced levels of the biomarkers α-SMA, FN and Col I (P<0.05). Meanwhile, concentrations of FN and Col I in cell culture supernatants also decreased (P<0.05). CONCLUSIONS:miR-145 modulates the EMT of HK-2 cells treated with TGF-β1, possibly by inhibition of the activation of TGF-β-dependent Smad signaling pathway.
Authors: Philip A Gregory; Andrew G Bert; Emily L Paterson; Simon C Barry; Anna Tsykin; Gelareh Farshid; Mathew A Vadas; Yeesim Khew-Goodall; Gregory J Goodall Journal: Nat Cell Biol Date: 2008-03-30 Impact factor: 28.824