The Roles And Mechanisms Investigation Of LCN2 And Ran In Liver

Backgroud:Hepatitis B virus(HBV),which mainly spreads by three paths,such as blood-borne infections,vertical and sexually transmitted infections,is a circular DNA belonging to hepadnaviridae and can lead to acute or chronic hepatitis,cirrhosis and hepatocellular carcinoma.Until now,HBV still cannot be eradicated and the strateges formulate on the basis of serological and biochemistrical biomarkers,such as HBV DNA?ALT?AST?HBsAg?HBcAg.However,there are so many factors can affect these indices that accurate disease status was misunderstood.Hence,it is essential to explore additional biomarkers that identify varying degrees of hepatitis B progression.The previous study using the gene microarray technique identified that four indices,LCN2,MIP2,Tim3 and iNOS,increased at the early phase of ConA-driven fulminant hepatitis.Besides,it was reported LCN2 was mainly produced by hepatocytes.In addition,the mechanism of Ran in hepatocellular carcinoma was still unknown.Concerned about these findings,the goals of our study were to assess the useful bilmarkers in recognizing varying stages of HBV-related liver disease and discovery the potential function and mechanism of LCN2 and Ran in liver.Part1:LCN2?Tim3?MIP2 and iNOS identified various stages of HBV-associated liver diseasesMethods:serum samples were collected and divided in six groups,such as health volunteers,HBV carrier,mild chronic hepatitis B patients,moderate chronic hepatitis B patients,severe chronic hepatitis B patients and fulminant hepatitis B patients and then LCN2?Tim3?MIP2 and iNOS was detected by ELISA.Meanwhile,the correlations were performed between all the lab indices.Besides,the roles of LCN2?Tim3?MIP2 and iNOS in distinguishing different stages of disease were deeply explored after grouping by ALT?TBIL and DBIL levels.Result;1.Along with disease progression,the levels of Tim3 and MIP2 gradually enhanced.The former was significantly different among all the groups(P<0.05).However,compared healthy controls with HBV carriers or mild chronic hepatitis B patients(P>0.05),there was no statistical difference of MIP2 levels.The expression levels of LCN2 and iNOS in patients were significantly elevated in severe chronic hepatitis B group and fulminant hepatitis B group respectively.2.There were significantly correlations between the above-mentioned indices and the conventional laboratory indices,the relationships were found between Tim-3 and MIP2(r = 0.498),ALB(r = 0.49),TBIL(r-0.487)and DBIL(r = 0.535).In addition,MIP2 was negatively associated with TBIL(r =-0.484)and ALB(r =-0.564),while it is positively corelated with ALT(r = 0.501),AST(r = 0.597),TBIL(r 0 0.584),DBIL(r = 0.632)and IBIL(r = 0.538).3.When ALT values were normal or not or when DBIL or TBIL levels were abnormal(P<0.05),MIP2 and Tim3 contribute to differentiate patients with fulminant hepatitis B from patients with moderate or severe chronic hepatitis B.Additionally,The MIP2 levels were significantly different among different phases of chronic hepatitis B patients when ALT values were normal or not or when TBIL was abnormal(P<0.05).Moreover,When ALT values were normal,there were statistical difference among healthy controls,HBV carriers and mild chronic hepatitis B patients.Part2:LCN2 exerted a protective role on IL-1?-induced hepatocytes stressMethods:the RNA interference technology was used to knockdown LCN2 expression level in Huh7 cells.Futhermore,the mRNA spectrum of differential expression genes between LCN2-siRNA group and negative control was achieved though microarray analysisResults:1.The mRNA and protein expression levels of LCN2,IL-6 and TNF-? in Huh7 cells were increased after IL-1? stimulation.2.Compared with negative control,the expression of IL-6 and TNF-? at mRNA and protein level in Huh7 cells were more enhanced by IL-1? stimulation in LCN2-knockdown group.3.Compared with negative control,there were 180 genes were upregulated and 268 genes were downregulated in the LCN2-silenced group and there are thirteen biological processes related to differential expression genesPart3:Ran played a role in autophagyMethods:After Ran was knockdown,tumor cell apoptosis,proliferation and invasion was explored through flow cytometry,cck8 and transwell experiments,respectively.In ·addition,WB and Immunofluorescence were used to detect the change of autophagy and autophagy-associated genes,such as sirtl,ATG5 and ATG7,when Ran was knockdownResults:1 The tumor cell proliferation and invasion was decreased after Ran was knockdown.2.Ran translocate from nuclear to cytoplasma during autophagy.3.There is no effect on apoptosis after Ran was knockdown.4.The expression level of sirtl was decreased after Ran was knockdown,while there was no change about ATG5 and ATG7.Conclusions:1.Our results demonstrated that ALT,DBIL or Tim3 levels could make a distinction among healthy subjects,HBV carriers,mild,moderate and severe chronic hepatitis B,and fulminant hepatitis B patients.Furthermore,comprehensive evaluation of LCN2 and MIP2 could accurately differentiate hepatitis B patients with various disease stages when the level of ALT was normal.2.Our data indicated that LCN2 upregulation protected hepatocytes from IL-1?-induced stress.In addition,microarray analysis demonstrated that LCN2 involved in protein metabolism,stress response,cell cycle and proliferation.3.Our results suggested Ran was likely to affect autophagy by modulating sirtl in HCC...