Transforming growth factor (TGF)-β1 may stimulate the activation of hepatic stellate cells (HSCs), resulting in the development of liver fibrosis. As micro RNA (miRNA)-122 is known to be associated with liver inflammation, its effects on the epithelial-mesenchymal transition (EMT) of HSCs through the inhibition of the TGF-β1/drosophila mothers against decapentaplegic protein 4 (Smad4) signaling pathway were investigated. The MTT assay was performed to explore the optimum TGF-β1 concentration suitable for HSC stimulation. Fluorescence microscopy was used to observe the transfection efficiency and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were used to observe gene and protein expression levels of α-smooth muscle actin (α-SMA), E-cadherin, N-cadherin and Smad4, respectively, in HSCs treated with TGF-β1 or TGF-β1 and miRNA-122. MTT assay results indicated that the concentration of 10 µg/l TGF-β1 was suitable for maximum growth and survival of HSCs. Notably, the mRNA expression levels of N-cadherin and α-SMA were significantly increased (each, P<0.05), but the expression levels of E-cadherin were decreased following 10 µg/l TGF-β1 treatment. Similar results were observed regarding the protein expression levels of N-cadherin, α-SMA and E-cadherin. Furthermore, the expression of F-actin was increased in the 10 µg/l TGF-β1 treated group compared with the 0 µg/l TGF-β1 treaded group and stretching of the muscle fiber filament was observed. miRNA-122 lentiviral vector transfection significantly decreased the mRNA expression of N-cadherin and increased the mRNA expression of E-cadherin in HSCs stimulated with TGF-β1, as evident from RT-qPCR results. Similar results were also observed regarding the protein expression levels of N-cadherin and E-cadherin. The expression levels of Smad4, the primary component of the TGF-β1 signaling pathway, were significantly lower in cells treated with TGF-β1 and miRNA-122 (P<0.01) compared those treated with TGF-β1. Thus, miRNA-122 may inhibit the activation and EMT of HSCs stimulated by TGF-β1.
Keywords: epithelial mesenchymal transition; liver fibrosis; microRNA-122; transforming growth factor-β1/smads signaling pathway.