The Effect Of MeCP2Regulate PTCH1Expression In HSC Activation And Proliferation

Hepatic fibrosis is a common element in many chronic liver diseases regardless ofthe exact etiology, causing the extracellular matrix (ECM) deposition in the liver,studies found that DNA methylation binding protein MeCP2play an important role inHSC activation and proliferation. Therefore, HSCs is activated by a variety ofpathological stimuli, thereby undergoing proliferation, differentiation to myofibroblasts,and production of various cytokines and ECM proteins. Thus, understanding thebiological processes of HSCs will provide novel insights into the underlyingmechanisms of hepatic fibrosis. DNA methylation is an important epigeneticmechanism, play a crucial role in hepatic fibrosis. TGF-β1plays a crucial role on theprocess of HSC activation in response to liver damage. Proliferation and activation ofcultured HSC with TGF-β1which are the main ECM producing cells in fibrosisprogression. This study mainly contains three sections, as belows:1. Shh、PTCH1、Gli1changes in SD rat hepatic fibrosisHepatic fibrosis was induced by CCl4. We successfully established the liverfibrosis model in SD rat, by CCl4administration at a dose of1.0ml/kg twice a week.Control group rats were established administration at a dose of1.0ml/kg olive oil at thesame time. After12weeks, the rats were fasted without water deprivation after the lastintragastric administration, and appropriate samples were collected after12h. After12 Weeks, HE and Masson staining to observe the histopathological changes under a lightmicroscopy. Shh, PTCH1, Gli1changes in rat liver tissues were analyzed by Westernblot, QRT-PCR respectively. Moreover, Shh、PTCH1、Gli1expression changes duringTGF-β1stimulation induced the activation of HSC in vitro. Therefore, the rats fromCCl4-induced hepatic fibrosis established well. The expression of PTCH1wasdown-regulated in liver fibrosis，but, Shh、Gli1expression was increased during hepaticfibrosis.2.5-AzadC and MeCP2inhibits HSC proliferation and activationTo reveal the effect of HSC-T6cells treated with siRNA-MeCP2and5-AzadC.HSC-T6cells were divided into three groups, treated with TGF-β15ng/ml. Moreover,added5-AzadC into DMEM media, and treated with two days, MTT assay analyzed theproliferation of HSC, HSC cell cycle was determinated by Flow cytometry, QRT-PCRanalyzed the levels of α-SMA， Col1A1mRNA in the HSC. QRT-PCR,immunohistochemical staining detected MeCP2, α-SMA expression in liver tissues.RNA interference inhibiting of MeCP2expression, LipofectamineTM2000serve asvectors and transfected into HSC-T6cells. Flow cytometry assay detected HSC cellcycle content. Treatment with HSC with5-AzadC reduced α-SMA,CollagenⅠexpression.5-AzadC and MeCP2-siRNA effectively inhibited the cellproliferation, indicating that5-AzadC and MeCP2play a key role in HSC proliferationand activation.3. MeCP2regulation of PTCH1expressionHepatic fibrosis results from the excessive secretion of matrix proteins by HSCs.Quiescent hepatic stellate cells transdifferentiation into myofibroblasts. Treatment ofHSCs with DNA methyltransferase inhibitor5-AzadC inhibit proliferation.5-AzadCalso upregulation of PTCH1expression in activated HSCs. To study the potentialmolecular mechanisms, we indicated that DNA methylation play a key role in liverfibrosis. It was shown that DNA methylation of PTCH1is associated with hepaticfibroblast activation and fibrosis. Silencing of MeCP2may increase the PTCH1 expressions in activated HSCs. This study indicated that MeCP2regulation of PTCH1expression through DNA methylation. In sum, our datas found that PTCH1may serveas a potential therapeutic target for hepatic fibroblast activation and fibrosis.