The Mechanisms Of LncRNA Gm40262 Regulating Liver Fibrosis Caused By Echinococcus Multilocularis Infection

Alveolar echinococcosis(AE)is a parasitic disease caused by Echinococcus multilocularis(E.multilocularis)infecting intermediate hosts.In the early stage of infection,the parasite stimulates the host liver and recruits a large number of immune cells.These immune cells then produce amount of cytokines,including transforming growth factor(TGF-β),which promote the activation of hepatic stellate cells(HSCs).The activated HSCs will transforme into myofibroblasts and produced a large amount of extracellular matrix,leading to liver fibrosis.Studies have found that long chain non-codingRNA(lncRNA)shows great potential in the treatment of liver fibrosis.Based on this,the following work was carried out in this study:By analyzing the pathological change of mice liver,it was found that E.multilocularis caused different degrees of liver fibrosis in 2(middle stage of infection)and 3 months(late stage of infection)post infection(2mpi and 3mpi).Based on this,by used high-throughput sequencing technology,the expression profiles of lncRNA and miRNA in HCs,HSCs and KCs at 2 mpi and 3 mpi were obtained.Through functional enrichment analysis of DElncRNAs,it was found that DElncRNAs in HSCs were mainly enriched in inflammation related pathways,such as Th1 and Th2 cell differentiation,Toll like receptor signal pathway and NF-κB signal pathway.And the lncRNA gm40262 enriched in HSCs was significantly upregulated after infection and it’s differential expression fold change is higher than HCs and KCs,which is associated with the fibrosis related molecule Col1α1 and α-SMA has the same expression trend,suggesting that gm40262 may be involved in E.multilocularis induced liver fibrosis.Further analysis revealed gm40262 had the binding capacity to miR-193b-5p,and miR-193b-5p had the binding capacity to TLR4.After infected,the TLR4 protein level were up-regulated and miR-193b-5p was down-regulated.Using hydatid cyst fluid(HCF)treated the HSCs,the same results was obtained.After over-expressed gm40262 in HSCs,which inhibited miR-193b-5p and increased TLR4 and IL-1β expression.Conversely,knocking down the expression of gm40262 increased miR-193b-5p and inhibited TLR4 and IL-1β expression.These manifests that gm40262 regulate inflammation of HSCs through gm40262-miR-193b-5p-TLR4 axis,which is beneficial to reduce fibrosis.In addition,HSC treated with HCF induced cell proliferation,while knockdown of gm40262 decreased HSC proliferation,which manifests that gm40262 could regulate the activation of HSCs cells.After bioinformatics prediction and double luciferase report experiment verification,it is found miR-193b-5p can target Col1α1 The expression of Col1α1 was inhibited after knocking down gm40262,which shows gm40262 could regulate fibrosis process through gm40262-miR-193b-5p-Col1α1 axis.In addition,the expression of fibrogenic cytokine TGF-β,Smad pathway protein molecules p-smad2 and p-smad3 and fibrosis marker proteins α-SMA and Col1α1 is also positively regulated by gm40262,indicating that gm40262 can activate TGF-β-Smad signal pathway for regulating ECM generation.Knockdown of gm40262 in the liver by packaging si-gm40262 using adeno-associated virus type 8can significantly alleviate liver fibrosis caused by E.multilocularis infection.The expression of TLR4,IL-1β,Col1α1 and α-SMA were significantly down-regulated by qRT-PCR and WB,which shows si-gm40262 has the potential to treat liver fibrosis.To sum up,the infection of E.multilocularis causes gm40262 significant up-regulated expression of HSCs,which can further regulate the expression of inflammatory factor and fibrosis related gene Col1α1 by targeting miR-193b-5p to accelerate the process of fibrosis.Therefore,knocking down gm40262 in the liver can inhibit liver fibrosis caused by E.multilocularis infection.The above research provides a theoretical basis for revealing the pathogenesis of E.multilocularis,interaction mechanism with host and further developing new drugs based on lncRNA.