Study On The Effect And Regulatory Mechanism Of Hotair In Liver Fibrosis

Liver fibrosis results from persistent liver jury,including viral hepatitis,alcohol abuse,metabolic diseases,autoimmune diseases,and cholestatic liver diseases.During fibrosis progression,inflammation and liver injury trigger complex cellular events that result in collagen deposition and the disruption of the normal liver architecture.Over the last two decades,sinusoidal resident hepatic stellate cells(HSCs)have been commonly recognized as the major source of extracellular matrix(ECM).In the normal liver,HSCs are quiescent,vitamin A-storing adipogenic cells,However,following a fibrogenic stimulus,HSCs undergo a complex activation process associated with morphological changes from a quiescent vitamin A-storing cell to that of an activated myofibroblast-like cell.HSC activation is also associated with a dramatic increase in the synthesis and deposition of ECM components,marked upregulation of ?-smooth muscle actin(?-SMA),collagen,tissue inhibitors of metalloproteinases(TIMP1)and desmin,production of profibrogenic cytokines/growth factors such as transforming growth factor-?(TGF-?),or platelet-derived growth factor(PDGF).Because HSC activation and liver fibrosis are orchestrated by the same signals,for example by growth factors such as TGF-?,the molecular mechanisms which exert global control of HSC activation and liver fibrosis is incompletely understood.Recent works from our group and from others implicated that lnc RNA plays an important role in determining HSC activation and liver fibrosis.Hox transcript antisense intergenic RNA(HOTAIR),with a length of 2158 bp,is located on chromosome 12q13.13,which has a functional role in trans-silencing.HOTAIR binds Polycomb repressive complex 2(PRC2),which is composed of EZH2, SUZ12,and EED,and recruits them to HOXD sites to promote coordinated H3K27 methylation for Hox gene silencing.HOTAIR expression levels were highly elevated in diverse types of cancers,including breast,colon and liver cancer,and HOTAIR was a negative prognostic factor for breast,colon and liver cancer patient survival.The expression level of HOTAIR in gastric cancer(GC)tissues was higher than that in adjacent noncancerous tissues.Expression level of HOTAIR was significantly correlated with lymph node metastasis and TNM stage.Furthermore,high expression level of HOTAIR was a predictor of poor over-all survival in liver cancer patients.However,the role of HOTAIR in liver fibrosis,especially the activation of HSC,has not been reported yet.So this study focuses on the role of Hotair in liver fibrosis,especially the role in HSC activation.The study intends to clarify the related molecular mechanism of liver fibrosis formation,providing a new target for preventing liver fibrosis,search for new liver fibrosis therapy,which will reduce the happening of liver cirrhosis and primary hepatocellular carcinoma(HCC).Thus,the significance of this study is high.Main content:1.The upregulation of Hotair expression in fibrotic liver tissue We first examined the expression of Hotair in CCl4-induced mouse models of liver fibrosis and in human hepatic fibrosis tissue.The expression of Hotair was detected by immunohistochemistry and RT-q PCR.In addition,human liver hemangioma tissue severed as a normal control group,while more than 2 cm adjacent to liver cancer tissue was identified as liver fibrosis tissue.The co-localization of Hotair and ?-SMA was observed by double immunofluorescence staining.The results showed that compared with the normal control group,the expression of Hotair was significantlyincreased in human liver fibrosis tissue and mouse liver fibrosis tissue,suggesting that Hotair mainly expressed in hepatic stellate cells.2.Effect of Hotair on HSCs Activation In vitro,LX-2 cells(activated HSC)were transfected with si-Hotair and Hotair plasmids.The proliferation of LX-2 cells was observed by CCK8 assay.The activation of LX-2cells was observed by RT-q PCR and western blot.The results showed that Hotair overexpression promoted the proliferation and activation of LX-2 cells compared with the control group,while knockdown of Hotair inhibited the proliferation and activation of LX-2 cells.3.Hotair acts as a ce RNA for miR-148 b to promote DNMT1 expression We further combined bioinformatics and luciferase reporter gene to study miR-148 b targeted inhibition Hotair and DNMT1.The effects of miR-148 b on Hotair expression and luciferase activity were observed by RT-q PCR and luciferase reporter assay.The results showed that compared with the control group,overexpression of miR-148 b resulted in the reduction of Hotair expression,while the inhibition of miR-148 b induced Hotair expression.In addition,miR-148 b significantly inhibited DNMT1 expression and luciferase activity.Luciferase,RT-q PCR and western blot results showed that compared with the control group,DNMT1 expression and luciferase activity were reversed in the presence of Hotair.4.Hotair promotes HSC activation by inhibiting MEG3 /p53 To further explore the molecular mechanisms involved in the effects Hotair on HSC activation,we used co-transfection,RT-q PCR and western blot to investigate whether MEG3 is involved in the functional effects of Hotair on HSC.Knockdown of Hotair up-regulates MEG3/p53 pathway in LX-2 cells,whereas overexpression of Hotair inhibits MEG3/p53 pathway.In addition,we co-transfected LX-2 cells with Hotair and MEG3 plasmids and observed the proliferation and activation of LX-2 by RT-q PCR and western blot.The results showed that compared with the control group,Hotair overexpression promoted the proliferation and activation of LX-2,whereas MEG3 overexpression reversed Hotair-mediated LX-2 proliferation and activation.5.Hotair / DNA inhibits MEG3 by PRC2 To investigate whether Hotair regulates gene expression through interactive histone modification(especially PRC2),we investigated how Hotair regulates MEG3 by using CHIP-PCR,RNA co-precipitation,RT-q PCR and western blot.Our results showed that PRC2 complexes EZH2 and SUZ12 bind to Hotair.In LX-2 cells,Hotair overexpression inhibited MEG3 expression,while si-EZH2 or si-SUZ12 partially eliminate Hotair-mediated MEG3 reduction.In addition,overexpression of Hotair significantly induced H3K27me3 enriched in the MEG3 promoter in LX-2 cells.Most importantly,RNase Ch IP results showed that RNase inhibitor treatment maximizes the recruitment of PRC2 to the MEG3 promoter region.Treatment with RNase H partially reduced the PRC2 to the MEG3 promoter region,whereas RNase A treatment had no significant inhibitory effect.