Long Non-coding RNA-LFAR1 Promotes The Pathogenesis Of Liver Fibrosis

Long noncoding RNAs(lncRNAs)have been demonstrated to be associated with a battery of biological functions and pathogenesis,including development,proliferation,apoptosis,survival,differentiation and carcinogenesis.The specific contribution oflnc RNAs in the pathogenesis of liver fibrosis has not been described.In this study,we determined the lnc RNA expression profile in the livers of fibrotic mice and normal mice by lnc RNA microarrays and real-time PCR.We identified a liver enriched lnc RNA-LFAR1(lnc RNA-Liver Fibrosis Associated lnc RNA1)through a detailed analysis of the expression of lnc RNAs in various tissuesand defined its expression profile,function and mechanism in liver fibrosis.This study may provide a novel mechanism and potential therapeutic approach for the treatment of hepatic fibrosis.Methods:1.For CCl4-induced liver fibrosis,the occurrence of liver fibrosis was determined by macroscopic examination,HE staining,Sirius red staining,immunofluorescent assay,immunohistochemistry(IHC),q RT-PCR,Western-Blot and the concentration of hydroxyproline in mouse livers.2.We then applied microarray analysis to compare lnc RNA and m RNA expression levels between fibrotic liver tissues and normal liver tissues,and performed differential screening analysis,GO analysis,KEGG pathway analysis,lnc RNA-m RNA coexpression analysis and predictive target genes of mi RNA analysis.3.To validate the findings of microarray analysis,we selected 10 lnc RNAs according to the fold change and the lnc RNA-m RNA coexpression network,10 m RNAs that are related to liver fibrosis from the results of microarray,and analyzed their expression using q RT-PCR in liver tissues,primary HSCs and hepatocytes.We discovered the existence of a liver enriched lnc RNA,and we named it Liver Fibrosis Associated lnc RNA1(lnc-LFAR1).4.We determined whether lnc-LFAR1 represents a noncoding gene,and performed cell fractionation followed by q RT-PCR and 5’-and 3’-rapid amplification of c DNA ends(RACE).5.We examined the expression of activation,fibrosis,and proliferation-related genes in primary HSCs infected with lentivirus of lnc-LFAR1 or lnc-LFAR1-sh RNA by confocal microscopy,q RT-PCR and Western-Blot.6.We examined the expression of fibrosis,apoptosis,and inflammation-related genes in primary HCs or AML12 cells infected with lentivirus of lnc-LFAR1 or lnc-LFAR1-sh RNA by confocal microscopy,FACS,q RT-PCR and Western-Blot.7.To explore the role of lnc-LFAR1 in liver fibrosis in vivo,lenti-sh LFAR1 or lenti-NC was intravenously injected into CCl4 and BDL-treated mice via the tail vein.8.We tested whether TGFβ might regulate the expression of lnc-LFAR1 through Smad2/3 by RNAi,Ch IP assay and luciferase reporter gene assay.9.In order to investigate whether lnc-LFAR1 affects liver fibrosis-related pathways including TGFβ,Notch,MAPK1,NF-κB pathways,we detected these pathways components or target genes in lnc-LFAR1 down-regulated or up-regulated Cells.10.We performed RIP and Ch IP assays to determine whether lnc-LFAR1 associates with Smad2/3.Results:1.Through a lnc RNA microarray in the livers of fibrotic mice and normal mice,we discovered the existence of a liver enriched lnc RNA-LFAR1.We found that,despite down-regulated lnc-LFAR1 level in whole liver lnc RNA extracted from the fibrotic mice,lnc-LFAR1 is specifically upregulated in HSCs during fibrogenesis.Moreover,TGFβ increased the level of lnc-LFAR1 in primary HSCs and hepatocytes.2.Lnc-LFAR1 promotes the expression of pro-fibrogenic genes and the activation of HSCs.3.Knockdown of lnc-LFAR1 reduces TGFβ-induced pro-fibrogenic gene expression and apoptosis in hepatocytes.4.Lnc-LFAR1 silencing attenuates CCl4-and BDL-induced liver fibrosis in vivo.5.Smad2/3 mediates TGFβ-induced lnc-LFAR1 expression.6.Lnc-LFAR1 up-regulates the expression of Smad2/3 and promotes its phosphorylation in liver fibrogenesis.7.Lnc-LFAR1 promotes liver fibrosis through activating Notch signaling.8.Lnc-LFAR1 promotes the association of TGF-βR1 with Smad2/3 and Smad2/3phosphorylation in the cytoplasm.9.Lnc-LFAR1 interacts with Smad2/3 to regulate target genes transcription in the nucleus.Conclusion:In this study,wedemonstrate that several lnc RNAs are specifically regulated in mouse models of liver fibrosis through a lnc RNA microarray analysis,leading to the identification of a liver enriched lnc-LFAR1,which could be regulated by TGFβ.Our data showed that lnc-LFAR1 functions as a positive regulator in liver fibrosis both in vitro and in vivo.Knockdown of lnc-LFAR1 significantly inhibited HSCs activation and reduced TGFβ-induced hepatocytes apoptosis.Moreover,lnc-LFAR1 silencing attenuated CCl4-and BDL-induced liver fibrosis in vivo.Mechanistically,our results indicated that lnc-LFAR1 promotes the association of TGF-βR1 with Smad2/3 and Smad2/3 phosphorylation in the cytoplasm.Furthermore,lnc-LFAR1 binds directly to Smad2/3,and this association activates both TGFβ and Notch pathways through directly regulating the transcription of TGFβ1,Smad2/3 and Notch signaling components such as Notch2,Notch3 and Hes1,revealing that TGFβ1/Smad2/3/lnc-LFAR1 pathway might provide apositive feedback loop that augments Smad2/3 response and a novel link connecting TGFβ1 signaling with Notchpathway.All these data support our conclusion that lnc-LFAR1 haspleiotropic effects on HSCs activation,hepatocytes apoptosis and liver fibrogenesis,thus providing support for its use as a potential target of fibrosis.