| ObjectiveLiver fibrosis is characterized by the excessive deposition of extracellular matrix.The main source of the extracellular matrix is from activated hepatic stellate cells. Some studieshave shown that the epithelial to mesenchymal transition (EMT) also plays an important role in liver fibrosis. EMT is a biological processin which epithelial lose their phenotypic characteristics and acquire typical features of mesenchymal cells such as fibroblasts.The microRNAs (miRs) are a class of non-coding small molecule RNAabout22to28nucleotides which play an important role in the post-transcriptional level and translational level of gene regulation.MiR-21has been shown toparticipate inmany physiological and pathological regulation.Our previous work has shown that miR-21plays an important role in hepatic stellate cell activating process. The purpose of this study is to explore the role and mechanism of miR-21in the EMT of the human hepatocytes.Methods1. The role ofTGF-β1in the EMT of the human hepatocytes.Human hepatocytes line QSG-7701cells were treated with lOng/ml TGF-β1.The changes in cell morphology were observed by microscope. The protein expression of epithelial marker protein and mesenchymal marker proteins was detected using western blot.2.TGF-β1stimulated miR-21expression. The expression of miR-21was detected by real-time RT-PCR after the treatment of human hepatocytes line QSG-7701cells with10ng/ml TGF-β1for1,3,5,7days.3.MiR-21mediated TGF-β1-induced EMT of human hepatocytes.After knocking down miR-21by transfecting with100nM miR-21inhibitor, QSG-7701cellswere stimulatedby TGF-β1for5days.24hours after transfection, miR-21expression was detected by real-time RT-PCR.The changes in cell morphology were observed by microscope.Western blot was used todetect the epithelial marker protein (E-cadherin) and the mesenchymal marker proteins (N-cadherin, vimentin).4.The role of microRNA-21in the EMT of the human hepatocytes. After up-regulation of miR-21expression by transfecting with100nMmiR-21mimics, real-time RT-PCR wasused todetect the expression of miR-2124hours after transfection, and western blot was used todetect the epithelial marker protein (E-cadherin) and the mesenchymal marker proteins (N-cadherin, vimentin)3days after transfection.5.PTEN/Akt pathway mediated the EMT of the human hepatocytes.(1)TGF-β1regulated the expression of PTEN protein:PTEN expression decreased after TGF-β1stimulation.(2)Aftertransfected with miR-21mimics and miR-21inhibitor, PTEN protein expression ofeach group wasdetectedbywestern blot.(3) After pretreated with a5μM Akt inhibitor (Akt inhibitor IV), the cells was stimulated by TGF-β1. Total Akt, Akt phosphorylation and the expressionsof epithelial marker protein (E-cadherin)and mesenchymal marker proteins (N-cadherin, vimentin) were detected by western blot.(4) After transfection of100nM of miR-21mimicsintohuman hepatocytes line QSG-7701cells,5μM Akt inhibitor (Akt inhibitor Ⅳ)was added into culture medium.Total Akt, Akt phosphorylation and the expressionsof epithelial marker protein (E-cadherin)and mesenchymal markers proteins(N-cadherin, vimentin) were detected by western blot.Results1.The role ofTGF-β1in the EMT of the human hepatocytes.(1) TGF-β1induced mesenchymal morphology in human hepatocytes which were characterized by slender and more tentaclescomparing withepithelial morphology.(2)The protein expression of epithelial marker,E-cadherin,wasdecreased afterTGF-1stimulation.(3) The protein expressions of mesenchymal marker (N-cadherin, vimentin) was increased afterTGF-β1stimulation.2. TGF-β1stimulated the expression of miR-21in hepatocytes,5days after TGF-β1stimulation.3. MiR-21mediated TGF-β1-induced EMT of human hepatocytes.(1)MiR-21expression significantly was declined,24hoursafter transfection of miR-21inhibitor;(2) Morphological changes:miR-21inhibitor group with tightarrangement showed more blunt-rounded shape cells and less tentacles comparing with the control group.(3) The expression of epithelial marker protein,E-cadherin, in miR-21inhibitor group was higher comparing with the control group.(4)The protein expressions of mesenchymal marker (N-cadherin, vimentin) in miR-21inhibitor group were lower than that of the control group.These results prompted that inhibition of miR-21blocked TGF-β1-induced hepatocyte EMT.4. MiR-21overexpression directly stimulatedthe EMT of human hepatocytes.(1)MiR-21expression increased greatly,24hours after miR-21mimicstransfection.(2) MiR-21mimics transfection group with loose arrangement showed slender and more fibroblast-like with more tentacles comparing with the control group.(3) The expression of epithelial marker protein,E-cadherin, in miR-21mimics group was lower than that of the control group.(4)The protein expressions of mesenchymal marker (N-cadherin, vimentin) in miR-21mimics group were higher than that of the control group.These results prompted that miR-21overexpression directly stimulatedthe EMT of human hepatocytes.5. PTEN/Akt pathway mediated the EMT of human hepatocytes.(1) TGF-β1regulated the expression of PTEN protein:the expression of PTEN was decreased after TGF-β1stimulation.(2) MiR-21regulated the expression of PTEN protein:the expression of PTEN declined after miR-21mimicstransfection; the expression of PTEN rised after miR-21inhibitortransfection.(3) Inhibition of Akt attenuated TGF-β1-induced EMT:the expression of epithelialmarker protein (E-cadherin) in Akt inhibitor+TGF-β1group was higher than that of the TGF-β1group, and the expression of mesenchymal markerproteins(N-cadherin, vimentin) in Akt inhibitor+TGF-β1group was lower than that of the TGF-β1group.(4)Inhibition of Akt attenuated miR-21-induced EMT:epithelial marker protein(E-cadherin) in Akt inhibitor+miR-21mimics group was higher than that of the miR-21mimics group, and the expression of mesenchymal markerproteins(N-cadherin, vimentin) in Akt inhibitor+miR-21mimics group was lower than that of the miR-21mimics group.ConclusionsMiR-21regulates the EMT of human hepatocytes which is mediated via PTEN/Akt pathway.
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