Molecular Mechanism And Clinical Significance Of Hepatitis B Virus X Gene EHBH2 Genotypes Affecting Cirrhosis And Hepatocellular Carcinoma Progress

Hepatitis B virus infection(HBV)is an important global health problem,especially in developing countries including my own.Although antiviral treatment against HBV can inhibit the replication of HBV,it still can not completely remove HBV.Some patients with chronic hepatitis B(CHB)develop progressive liver fibrosis and gradually lead to cirrhosis and even primary hepatocellular carcinoma(short for liver cancer,HCC).In our country,due to the extensive HBV infection and the non-standardization of antiviral therapy,cirrhosis and hepatocellular carcinoma occur in a high proportion.Cirrhosis and liver cancer caused by chronic HBV infection seriously threaten the life and health of our people,which is a major issue that needs to be addressed in our 13 th Five-Year Plan.As one of HBV four open reading frames,HBV X gene can encode HBx protein with a molecular weight of 17 KD.A large number of studies have confirmed that HBx protein plays a crucial role in the development of liver cirrhosis and HCC and is a particularly important viral protein.There are many variations in HBx DNA due to inaccurate reverse transcription and lack of proofreading in HBV replication.In a previous study,we investigated genotypes of HBx in a large HCC cohort of tumor and nontumor tissues and found that a genotype that carries multiple variant locus,HBx-EHBH2(HBx-E2).Prognostic analysis shows that patients with HBx-E2 have a better prognosis.In addition,we also preliminary study of the biological function of HBx-E2 in hepatoma cells.However,the role of HBx-E2 in predicting the prognosis of HCC patients and its significance in clinical transformation also need to verify in multi-center cohort.The function of HBx-E2 in HCC occurrence and development,as well as the specific molecular mechanisms need to explore further.Furthermore,the correlation between HBx-E2 and clinicopathological parameters showed that HBx-E2 was associated with the loss of liver cirrhosis.Thus,HBx-E2 may played a different role with other genotypes in the process of cirrhosis and need further study.Therefore,we conducted the research on the above scientific problems.Section One The molecular mechanism of HBx gene EHBH2 genotype affecting the progression of hepatocellular carcinomaPurpose: To clarify the role of HBx-E2 genotypes in preoperatively predicting the prognosis of hepatocellular carcinoma patients.To determine the biological function of HBx-E2 in the development of HCC and related molecular mechanisms.Method: HBx genotypes were detected in cohorts of patients with HCC from different regions.Prognostic analysis and clinicopathological analysis of HBx-E2 were also performed.To construct multiple HCC cell lines and liver cell lines expressing multiple HBx genotypes,the effects of HBx-E2 on cell proliferation were determined by cell counting kit 8(CCK8),colony formation and flow cytometry.The recombinant HBx protein and protein chip were used to screen the difference of HBx-E2 and wild-type HBx protein binding protein in vitro,and the molecular mechanism was explored by co-immunoprecipitation and confocal laser scanning microscopy.Result: HBx-E2 was associated with a better prognosis in the tumor and nontumor tissue of HCC cohort(n = 171)from different regions and in the serum cohort(n = 168).Correlation analysis of clinical pathological indicators showed that HBx-E2 was associated with a lack of cirrhosis and a small diameter of the tumor.According to the HBx genotyping and Barcelona Clinic Liver Cancer(BCLC)staging,the stratified analysis was performed in HCC tissue cohort and serum cohort.The results showed that HBx-E2 could be used as a molecular marker to predict the prognosis of patients with BCLC B stage before surgery.In vitro and in vivo experiments demonstrated that HBx-E2 loses the ability to promote the proliferation of hepatoma cells and hepatocytes.Protein microarray and coimmunoprecipitation experiments found that HBx-E2 loses the ability to bind to JAK1 and does not activate STAT3 and STAT5.Upadacitinib,an inhibitor of JAK1,inhibits the proliferation of other genotypes except HBx-E2.Conclusion: HBx-E2 can be used as a molecular marker to preoperatively predict the prognosis of patients with BCLC B stage.HBx-E2 loses its ability to promote the proliferation of hepatoma cells and hepatocytes,and can not activate the JAK1/STATs pathway.JAK1 is a potential therapeutic target for HBV-related HCC patients with some HBx genotypes.Section Two The role and molecular mechanism of HBx gene EHBH2 genotype in liver cirrhosisPurpose: HBx promotes the progress of hepatic cirrhosis by promoting the secretion of cytokines by hepatocytes and leading to the activation of hepatic stellate cells(HSCs)in a paracrine manner.This part of the study mainly focus on the different activation of HSCs by hepatocytes with various HBx genotypes,and clear the role and molecular mechanism of HBx-E2 genotype in HBV-related cirrhosis progress.Method: HSCs were stimulated by the culture supernatant of various HBx genotype hepatocyte,and the activation of HSCs was detected by real-time PCR,western blot and immunofluorescence.To detecte the cytokines that led to the difference of HSCs activation,the difference of cytokines in the supernatant of hepatocytes expressing HBx genotypes was detected by ELISA.By adding neutralizing antibodies,recombinant cytokines,related receptor inhibitors to further prove the target cytokine on HSC activation.The use of technology such as western blot clear HBx genotypes on the regulation of the target cytokine.Result: The degree of cirrhosis in nontumor tissues of HCC patients carrying HBx-E2 genotype was significantly lower than those in other genotype carriers.The ability of hepatocyte expressing HBx-E2 genotype to activate HSCs was significantly weaker than those of other genotypes,and its ability to promote HSCs proliferation and migration was also significantly weaker than those of other genotypes.The difference in HSC activation between hepatocytes expressing various HBx genotypes is due to differences in the secretion of VEGF.Fruquintinib,a VEGF receptor inhibitor,can eliminate the difference of HSCs activation in various HBx genotype hepatocyte.For different HBx genotypes,TGF-β receptor inhibitor A83-01 alone or in combination with Fruquintinib could inhibit HBxactivated HSCs.Inhibition of p-STAT3 activation in other genotype hepatocytes can inhibit VEGF secretion.Conclusion: HBx-E2 failed to activate the JAK1/STAT3 pathway and thus lost its role in promoting VEGF secretion,leading to a significantly weaker ability of hepatocytes expressing HBx-E2 genotype to activate HSCs than other genotypes.TGF-beta receptor inhibitors and VEGF receptor inhibitors may serve as potential clinical targeted drug for blocking the progression of liver fibrosis and cirrhosis.