Over-expression Of Lnc-Scarna10 Induces Liver Fibrosis

A variety of chronic liver diseases such as viral hepatitis,schistosomiasis,autoimmune liver disease can cause liver fibrosis.As the disease progressed it results in cirrhosis,eventually leading to organ failure and death.In recent years,with the extensive research on long noncoding RNAs(lnc RNAs),it has revealed that lnc RNAs are widely involved in various biological functions and pathological processes relating with proliferation,inflammation,necrosis.However,few of lnc RNAs have been identified in the progression of liver fibrosis.In this study,we aim to select the abnormally expressed lnc RNAs that has species homology and reveal its function and mechanism in hepatic fibrosis.The expression level of SCARNA10 was increased in the serum and liver samples from patients with advanced hepatic fibrosis.Furthermore,the correlation between lnc RNAs and liver fibrosis in blood samples and tissue samples from patients with liver fibrosis and cirrhosis was confirmed,providing a proof of SCARNA10 as a potential diagnosis marker;its role in liver fibrosis and its mechanism were revealed in vitro and in vivo,providing a proof of SCARNA10 as a possible therapeutic target against liver fibrosis.Methods:1.Liver fibrosis mice model: the occurrence of liver fibrosis was determined by macroscopic examination,hydroxyproline content determination technique,HE staining and Sirius red staining,Western blot,q RT-PCR and immunohistochemistry in mouse liver.2.We then applied microarray analysis to select ten lncRNAs that are differerently expressed between fibrotic liver tissues and normal liver tissues.Subsequently we performed KEGG Pathway analysis,GO analysis,homology analysis and neighboring gene analysis to selecte a lnc RNA which significantly increased and has species homology,lnc-SCARNA10(ENSMUST00000158992).3.We validate the sequence of lnc-Scarna10 in both human and mice by 5'-and 3'-rapid amplification of c DNA ends(RACE).Subsequently we identified the location of lnc-Scarna10 in cell nuclear and cytoplasm.4.The expression of lnc-Scarna10 was detected in mouse liver cell line AML12 cells and mouse primary hepatocytes HCs;the expression of lnc-SCARNA10 was detected in blood samples and liver tissues of normal human,liver fibrosis patients and liver cirrhosis patients,confirmming that lnc-SCARNA10 has potential as a clinical diagnostic marker for liver fibrosis;the expression of lnc-Scarna10 and profibrotic related genes were tested as well as inflammatory genes in primary HCs derived from normal and hepatic fibrosis mice;the expression of lnc-Scarna10,profibrotic-related genes and pro-inflammatory related genes and apoptosis-related genes were detected in TGF?-treated mouse liver cell line AML12 cells and mouse primary HCs.5.Primary HCs or AML12 cells were transfected with of over-expression or knockdown lnc-Scarna10 lentivirus,then we examined the expression of lnc-Scarna10,profibrotic-related genes,inflammatory genes and apoptosis genes by q RT-PCR and Western blot.6.Lenti-sh Scarna10 or lenti-NC was intravenously injected into mice via the tail vein which have been opretated bile duct ligation to explore the role of lnc-Scarna10 in liver fibrosis in vivo.7.We detected the expression of TGF? pathways components and target gene in lnc-Scarna10 up-regulated or down-regulated hepatocytes to investigate whether lnc-Scarna10 affects liver fibrosis via related pathways.Results:1.In this study we screen the lnc-Scarna10 that is highly expressed during liver fibrosis and has homology in human and mice;lnc-Scarna10 is significantly up-regulated in primary HCs of liver fibrosis mice that are cultured in vitro.Moreover,TGF? was used to treat mouse liver cell line AML12 cells and mice primary HCs,the level of lnc-Scarna10 is significantly increased.2.lnc-SCARNA10 is up-regulated in serum and liver tissues of patients with liver fibrosis and cirrhosis compared with that of normal people,and the expression is higher in serum and liver tissues of cirrhosis patients than that of liver fibrosis patients.3.Knockdown of lnc-Scarna10 reduces TGF?-induced pro-inflammatory genes expression,pro-fibrogenic gene expression and apoptosis in hepatocytes.4.Knockdown of lnc-Scarna10 attenuates BDL-induced liver fibrosis in vivo.Also,we report that lnc-Scarna10 promotes liver fibrosis both in vitro and in vivo.Moreover,lnc-Scarna10 promotes liver fibrosis by promoting apoptosis of HCs and expression of pro-fibrogenic genes and pro-inflammatory genes.5.Knockdown of lnc-Scarna10 inhibited the expression of TGF?,SMAD2/3,p-SMAD2/3,KLF6 and other genes related to liver fibrosis in the TGF?signaling pathway.Conclusion:In this study we screen the lnc-Scarna10 that is highly expressed during liver fibrosis and has homology in human and mice.We confirmed that lnc-Scarna10 not only promotes the apoptosis of hepatocytes,but also promotes the expression of pro-fibrogenic and pro-inflammation genes both in vitro and in vivo.Mechanistically,Our results demonstrate that Scarna10 functions as a novel regulator of TGF?signaling,induces HCs apoptosis and promotes liver fibrogenesis.Furthermore,lnc-SCARNA10 is up-regulated in serum and liver tissues of patients with liver fibrosis and cirrhosis compared with that of healthy people,suggesting that lnc-SCARNA10 has the potential to be a clinical diagnostic marker for liver fibrosis and provide a novel treatment target for liver fibrosis,which has important value of application in clinic.