MicroRNA-146a Modulates TGF-beta1-induced Hepatic Stellate Cell Proliferation By Smad4

Hepatic fibrosis is a wound-healing process that occurs when the liver is injuredchronically. Activation of hepatic stellate cells (HSC) plays a pivotal role in thedevelopment of hepatic fibrosis and is responsible for the excess production ofextracellular matrix (ECM) components. Transforming growth factor-β1(TGF-β1) isconsidered to be the main stimuli factor responsible for the activation HSC.MicroRNAs (miRNAs) have recently been shown to regulate cell proliferation,differentiation, and apoptosis and so on. MiR-146a is the first found to be associatedwith the immune function, but the involvement of miR-146a and the roles inTGF-β1-induced HSC activation remains largely unknown. This study is to explorethe abnormal expression and molecular mechanisms of miR-146a in HSC and hepaticfibrosis and provide a novel therapeutic target for treating hepatic fibrosis.Liver fibrosis was generated by12-weeks treatment of adult maleSprague-Dawley (200-220g) rats with CCl4(CCl4/olive oil,1:1(vol/vol) per kg bodyweight by intraperitoneal injection twice weekly) as previously described. Controlanimals were treated intraperitoneally with1ml of olive oil/kg body weight at sametime intervals. After12-Weeks, the pathological and histological Masson collagendyeing were also detected and the expression of α-SMA in rat liver tissues wasmeasured by immunohistochemical staining. MiR-146a expression was analyzed indifferent concentrations of TGF-β1-treated HSC and CCl4-induced liver fibrotictissues by one-step real-time quantitative PCR. To investigate the effects ofoverexpression of miR-146a on TGF-β1-induced HSC proliferation by transfectingwith the miR-146a mimics into HSC through using LipofectamineTM2000according to the manufacturer’s instructions at a final concentration of60nM for24h or48h.The culture medium was changed6h after transfection, and TGF-β1was added at aconcentration of10ng/ml. Furthermore, the effects of miR-146a on theTGF-β1-induced HSC proliferation were investigated by MTT assay, cell cycleanalysis, and flow cytometry. Next, Smad4was found to be a target for miRNA-146aby using bioinformatics approaches. The expression of α-smooth muscle actin(α-SMA), a marker for HSC activation and Smad4were analyzed bySemi-quantitative reverse transcription-polymerase chain reaction (RT-PCR),real-time qPCR, and western blots in TGF-β1-treated HSC.In our study, we revealed that the expression of miR-146a was downregulated inHSC in response to TGF-β1stimulation (0,5,10, and15ng/ml) in dose-dependentmanner. Moreover, miR-146a was also downregulated in CCl4-induced liver fibrotictissues. In addition, The HSC transfected with miR-146a mimics(60nM) significantlydecreased TGF-β1-induced α-smooth muscle actin (α-SMA) mRNA and proteinexpression compared with the control. Furthermore, Overexpression of miR-146asignificantly suppressed TGF-β-induced HSC proliferation(P<0.05), and increased theproportion of apoptotic cells. Bioinformatics analyses predict that Smad4is thepotential target of miR-146a. MiR-146a overexpression in TGF-β1-treated HSC didnot decrease Smad4mRNA levels, but significantly reduced Smad4proteinexpression. MiR-146a regulated Smad4gene expression at the posttranscriptionallevel. These studies demonstrated that miR-146a modulates TGF-β1-induced HSCproliferation by inhibiting Smad4at the posttranscriptional level and may providemolecular mechansims for the development of liver fibrosis.