A Preliminary Study Of The Up-regulated LncRNA By HCV Infection And HCV Mouse Infection Model Optimization

Backgroud:Hepatitis C virus(HCV)is one of important pathogens leading to human liver diseases.According to WHO2017,about 71 million people worldwide have chronic infection with HCV,with an annual increase of 399000,mainly through blood spread.Human acute HCV infection is often easy to be developed into chronic hepatitis if without proper treatment,and then into liver fibrosis,liver cirrhosis,even liver cancer.Although the current direct-acting antiviral agents(DAA)drugs for the treatment of hepatitis C patients with a cure rate of up to 95%,but with the extremely expensive cost,poor compliance,and even viral resistance.Recent studies have shown that long non-coding RNAs(lncRNAs)play important roles in regulating cell metabolism,proliferation and differentiation,tumor development,disease diagnosis and prognosis,and potential targets of drugs.The regulation of the occurrence,development,outcome and prognosis of hepatocellular carcinoma is closely related to lncRNA,but unfortunately little is known about the role of lncRNA in the HCV-infected hepatocellular carcinoma cell line Huh7.5.1.For many years the animal model for HCV infection is limited to chimpanzees.In recent years,humanized HCV receptor transgenic mice have been developed to support HCV infection and full life cycle of replication,but this infection model also has disadvantgages with low successful infection efficacy.Objective:To study the differential expressions of long noncoding RNAs(lncRNAs)in human Huh7.5.1 hepatoma cell model after infection with HCV and their effects on proliferation,migration,invasion and cycle-related gene expression of Huh7.5.1 cells,which is helpful for better understanding the mechanism of HCV infection and liver cancer cell proliferation.These abnormally expressed lncRNA could be the potential tumor biomarkers and drug targets,which would be the candidates for diagnosis and treatments of HCV infection and hepatocellular carcinoma.In this study,we have also tested whether NK GMlblock antibody,IL10 and ApoE3 could improve the HCV infection efficacy on the basis of genetically humanized ICRTg4R+ mouse.Methods:The expressions of IncRNAs were analyzed with microarray technology,and the differentially expressed lncRNAs were verified by quantitative real-time PCR(RT-qPCR).CCK8 and Ki67 cell proliferation assays were used to detect the effects of lncRNAs on cell proliferation.The mRNA expressions of cell cycle related genes cyclin B1/D1/E1 were analyzed by RT-qPCR.Finally,Transwell experiments were used to detect the effects of cell migration and invasion.NK GMlblock antibody,IL10 and ApoE3 were injected by tail vein into ICRTg4R+ mice,respectively,at one day before HCV challenge.Then,the viral RNA and proteins levels from each mouse serum and liver tissue were detected at different time points.Results:Compared with untreated cells,the expression level of 3563 lncRNAs was up-regulated and the expression level of 5639 lncRNAs was down-regulated in Huh7.5.1 cell after HCV infection 3 days.17 abvious changing lncRNAs were confirmed by qRT-PCR,two of which were selected for futher investigation.The expression of lncRP11-288L9.1 was up-regulated,and the expression of lncADAM20P1 was down-regulated in HCV-infected cells.Silencing lncRNARP11-288L9.1 could inhibit the expression of cyclin Bl/D1/E1 and inhibit the proliferation of Huh7.5.1 cells,affect the cell migration and invasion ability.We aslo found that exogenous injection of GM1,ApoE3,and IL10 could promote HCV infection with enhanced viral loads in mouse sera and liver tissues.Conclusion:In summary,we found that the upregulation of expression of lncRP11-288L9.1 by HCV induces proliferation,migration,invasion and cycle-related gene expression of Huh7.5.1 cells.We aslo found that exogenous injection of GM1,ApoE3,and IL10 could promote HCV infection with enhanced viral loads in mouse sera and liver tissues.