Study Of LncRNA Promoting Fibrosis In Patients With Chronic Hepatitis B Treated By Nucleoside Analogs

Background:Long-term hepatitis B virus infection may have adverse consequences:including liver decompensation,cirrhosis,and hepatocellular carcinoma(HCC).As a global health problem,Chronic hepatitis B virus(HBV)infection is still lacking treatment measures,and nearly 1 million deaths are caused by HBV infection.In 2016,there were approximately 292 million infected patients worldwide,and the distribution of infected patients were mainly located in Asia and Africa(63.8%in Asia and 27.6%in Africa).Among them,the number of patients with chronic hepatitis B infection in China has reached 100 million.The economic and social burdens caused by it cannot be underestimated.During the chronic infection of hepatitis B virus,chronic HBV infection is a dynamic process of interactions between HBV,hepatocytes and the immune system of the host that leads to different phases of infection.The HBV infection process is mainly divided into the following four stages:immune tolerance period(chronic HBV carrier state),immune clearance period(HBe Ag positive CHB),immune control period(inactive HBs Ag carrier state)and reactivation period.In order to achieve the goal of“Total Elimination of Hepatitis B Virus Infection by 2030”proposed by the World Health Assembly,it is necessary to provide accurate treatment for patients with chronic hepatitis B virus in line with different stages of infection.Chronic infections are responsible for constant inflammatory stimulation.Immune cells activate the proliferation and differentiation of hepatic stellate cells through various ways,leading to the deposition of extracellular matrix.Extracellular matrix deposition is a major pathological change that causes fibrous scar formation and intrahepatic structural reconstruction in the liver.The use of anti-nucleotide drugs with hepatitis B vaccination and other treatment programs have reduced the amount of DNA virus copies in hepatitis B patients and improved their liver function.However,patients who have normal liver function after treatment still have the risk of liver fibrosis and liver cancer.Therefore,the purpose of this study is to investigate whether the outbreak of hepatitis affects the level of DNA transcription in asymptomatic hepatitis B carriers(ASC)and patients with normal liver function(CHB),which leads to a difference in prognosis between them.Methods:Peripheral blood mononuclear cells(PBMCs)from ASC patients and treated CHB patients were isolated and total RNA was extracted.Arraystar(human Lnc RNA/m RNA expression microarray)chip sequencing was used to analyze the differential m RNA and long non-coding RNA(lnc RNA)between the two groups of patients.Then KEGG was used to analyze the differential genes and the pathways related to liver fibrosis and liver cancer development.Based on the analysis results,the up-regulated m RNAs and related lnc RNAs in treated CHB patients were screened,and the sample size was further expanded for verification.PBMCs were sorted by using magnetic beads to further determine the source of lnc RNA.At the same time,the cytoplasmic ratio of lnc RNA was identified by separating cytoplasm and nucleus RNA,and Thp1(human peripheral blood mononuclear cell line)cells were stimulated by virus to determine the association between HBV virus and lnc RNA.According to the lnc RNA localization and nuclear cytoplasmic distribution,we designed the si RNA(Small interfering RNA)and lentiviral vector to knock down and over-express the target lnc RNA in macrophages.ELISA was used to detect the changes of TGF-?1 in the cell supernatant.Finally,by co-culturing Thp1 cells with Lx-2(human hepatic stellate cells),q-PCR and Western Blot were used to observe the effect of macrophages on hepatic stellate cells.At the same time,the expression of?-SMA(smooth muscle actin)and Collagen I(Collagen protease I)in LX-2 cells were detected by immunofluorescence staining technology.The fluorescence intensity of the cells was detected and counted under a confocal microscope.Results:By analyzing the results of the sequencing chip,we screened 1254 differentially expressed m RNAs,of which 630 genes were up-regulated in CHB and 624 genes were down-regulated.KEGG analysis showed that the differential genes in CHB were significantly enriched in the TGF-?pathway.The q-PCR method to expand the sample size verification showed that the expression of Smad4 and TGF-?1 was significantly increased in the CHB patient group.In plasma,the levels of TGF-?1 in patients with CHB were significantly higher than those in the ASC group.At the same time,intracellular Smad4 levels in PBMC of CHB group were significantly higher than those of ASC group.According to big data analysis of bioinformatics,we screened lnc RNAs associated with Smad4.The lnc RNA(ENST00000519726)is highly expressed in the cytoplasm of CD14~+cells in patients with CHB,and we named it HEIM.After HBV virus stimulation,the expression of Smad4 and TGF-?1 increased with the increase of HEIM expression.In addition,we further verified in the macrophage cell line Thp1 that knockdown expression of HEIM can significantly reduce the m RNA and protein levels of Smad4and TGF-?1 in cells.At the same time,it can significantly reduce the level of TGF-?1in the supernatant.Lentivirus was used to infect macrophages to construct a Thp1 cell line stably overexpressing lnc RNA.According to the results of q-PCR and Western blot,overexpression of HEIM can up-regulate the m RNA levels of Smad4 and TGF-?1 and thus promote their protein expression levels.Co-culture the treated Thp1 with LX-2,and Thp1 cells with low expression of HEIM could down-regulate the expression of Collagen I and?-SMA of LX-2.At the same time,Thp1 overexpressing HEIM could significantly increase the expression of Collagen I and?-SMA of LX-2,and confocal microscopy revealed significant increase in fluorescence intensity of Collagen I and?-SMA in LX-2.Conclusion:The above research shows that the abnormal expression of lnc RNA is one of the factors affecting the prognosis of patients with hepatitis.Using q-PCR technology,we screened lnc RNAs that were highly expressed in treated patients.The lnc RNA is highly expressed in peripheral blood CD14~+macrophages,and is mainly located in the cytoplasm of the cells.Highly expressed lnc RNA promotes the secretion of cytokine TGF-?,which in turn activates liver stellate cells LX-2.Compared with chronic hepatitis B carriers with normal liver function,patients with chronic hepatitis B who have normal liver function after antiviral therapy have a higher risk of developing fibrosis.In summary,in the process of long-term repeated liver damage caused by hepatitis B virus,even if liver function returns to normal after drug treatment,patients still need to actively perform antifibrotic therapy.The use of RNA interference technology to regulate the HEIM level of macrophages in treated patients can be one of the measures to improve the prognosis of patients with hepatitis B in the future.