Objective:To explore the relationship and dynamic changes between virological markers and hepatic pathological damage due to host anti-hepatitis B virus (HBV) immunity in the natural course of disease in chronic HBV infected patients.Methods:Two hundred and thirty-eight adult chronic HBV-infected patients who underwent liver biopsy from January 2016 to June 2022 in Taizhou Hospital, Zhejiang Province, were retrospectively selected. General clinical data such as age, gender, platelets, ALT, AST, albumin, HBV DNA, qHBsAg, HBeAg, and liver pathology diagnostic indexes such as the grade of liver necroinflammation and liver fibrotic stages of the patients were collected. The patients were grouped according to HBeAg status, and subgrouped according to different grades of liver necroinflammation and different HBV DNA loads. Statistical analyses were performed to compare the differences in HBV virologic marker levels between the groups, and the correlation between them and the indicators of hepatic inflammatory injury, such as ALT,AST, and the grade of liver necroinflammation in the patients.Results:The levels of HBV virological markers in HBeAg-positive patients with moderate or higher liver necroinflammatory grade (G≥2) were significantly lower than those with mild (no) liver necroinflammatory grade (G < 2) ( P < 0.01); whereas the opposite trend was observed in HBeAg-negative patients, with the levels of HBV DNA, and qHBsAg in the G≥2 subgroup being significantly higher than those in the G < 2 subgroup ( P < 0.01). Correspondingly, HBV DNA level and qHBsAg showed weak to moderately strong negative correlation with liver necroinflammatory grade and AST which was an indicator of hepatic inflammatory injury in HBeAg-positive patients ( P < 0.05); whereas in HBeAg-negative patients, they showed weak to moderately strong positive correlation with hepatic inflammatory activity and ALT, AST ( P < 0.001), in which qHBsAg showed only a weak positive correlation with patients' liver necroinflammatory grade ( P = 0.003). Further subgroup analyses of HBeAg-positive patients according to whether the HBV DNA level was > 2×10 6 IU/ml showed weak to moderate negative correlations between HBV virological markers and liver necroinflammatory grade as well as ALT and AST in the subgroup of patients with HBV DNA > 2×10 6 IU/ml ( P < 0.05); however, the negative correlation disappeared in patients who were still HBeAg positive and had HBV DNA ≤ 2×10 6 IU/ml. Moreover, HBV DNA and ALT, HBeAg and AST showed moderate positive correlation ( P < 0.05). Conclusion:We speculate that the activation of host anti-HBV immunity can efficiently inhibit HBV replication by targeting the infected hepatocytes, but only in the early phase of disease progression in HBeAg positive patients with HBV DNA high (> 2×10 6 IU/ml).

Objective:To evaluate the safety of discontinuing nucleoside/nucleoside analogue (NAs) therapy in patients with compensated hepatitis B cirrhosis after HBsAg negative conversion.Methods:A total of 3 783 patients with hepatitis B cirrhosis in compensated stage were treated with NAs at Taizhou Hospital, Taizhou Municipal Hospital and Taizhou Enze Hospital from January 2008 to December 2020. The clinical data and laboratory tests results of 85 patients with HBsAg negative conversion were retrospectively analyzed, including 36 cases discontinued the drug, and 49 continued to use drug. Chi-square test and rank-sum test were used for data analysis.Results:During the 24 and 48 months of follow-up, the ALT levels were within the normal range in both groups. There were no significant differences in positive rates of anti-HBs and HBeAg ( χ2=0.75, 0.39 and 0.90, P=0.78 0.84 and 0.34; χ2=0.40, 0.00 and 0.00, P=0.84, 1.00 and 1.00) between two groups. After 48 months of follow-up, 2 cases of primary liver cancer occurred in the discontinuation group and no primary liver cancer occurred in the continuation group ( χ2=0.89, P=0.34). Throughout the follow-up, HBsAg remained negative and HBV DNA load was below the lower limit of detection in both groups. Conclusions:Discontinuation of NAs can be considered after the HBsAg negative conversion in patients with compensated hepatitis B cirrhosis.

Liver cancer has a high degree of heterogeneity. The molecular characteristics of tumor in different patients, and even in the same liver cancer tissues can be significantly different. Current clinical and pathological classifications cannot accurately evaluate the heterogeneity of liver cancer. With recent developments in genomics, proteomics and metabonomics of liver cancer, molecular classification of liver cancer has made some progress. Intensive research on the immune microenvironment of liver cancer has promoted the recognition of immunological classification of liver cancer. However, none of these classifications can be translated into treatment strategies to guide clinical practice. Immunotherapy combined with antiangiogenic targeted drugs have achieved a great breakthrough in treating patients with advanced liver cancer. It is becoming established as the first-line treatment for patients with advanced, or even intermediate stage of liver cancer. In this review, recent research progress on` molecular and immunophenotyping and immune checkpoint inhibitors combined with antiangiogenesis inbibitors in advanced liver cancer are elaborated.

Objective To assess the diagnostic value of serum soluble mannose receptor (sMR) for hepatic fibrosis in patients with chronic hepatitis B (CHB).Methods Fifty patients with CHB undergoing liver biopsy in the Department of Infectious Diseases , Taizhou Hospital of Zhejiang Province from November 2016 to October 2018 were enrolled, including 28 males and 22 females.According to the stage of liver fibrosis, there were 15 cases without fibrosis (S0 group), 12 cases of mild fibrosis (S1-2 group), and 15 cases of moderate-severe fibrosis ( S3-4 group).Twenty healthy subjects (12 males and 8 females) were recruited as controls.Enzyme linked immunosorbent assay (ELISA) was used to detect the serum hyaluronic acid (HA), laminin (LN), procollagen type ⅢN-terminal peptide (PⅢP), collagen type IV (CIV) and sMR in all groups.One-way ANOVA, Spearman correlation analysis and receiver operating characteristic (ROC) curve were used to analyze the data.Results The serum levels of sMR, HA, LN, CIV and PⅢP in S3-4 group were significantly higher than those in S 0 group ( t＝10.20, 4.69, 8.94, 2.35 and 4.34, respectively; all P＜0.05) and S1-2 group (t＝5.77, 4.23, 7.88, 2.71 and 3.43, respectively; all P＜0.05); and serum sMR level in S1-2 group was higher than that in S0 group ( t ＝6.23, P ＜0.05). Spearman rank correlation demonstrated that serum sMR level was positively correlated with the degree of liver fibrosis (r＝0.860, P＜0.01).ROC curve analysis showed that when 228.69 ng／mL was taken as cut-off value of sMR, its specificity and sensitivity for diagnosis of hepatic fibrosis were 93.3%and 88.6%, respectively.The diagnostic efficacy of sMR was significantly better than that of HA , LN, CIV and PⅢP (Z＝3.179, 3.467, 5.241 and 3.567, respectively; all P＜0.05).When 345.80 ng／mL was taken as cut-off value of sMR, the specificity and sensitivity for diagnosis of moderate to severe hepatic fibrosis were 85.7%and 86.7%, respectively; and its diagnostic efficacy was better than that of HA , CIV and PⅢP (Z＝2.253, 2.475 and 2.092, all P ＜0.05).Conclusion Serum sMR level is associated with the progression of liver fibrosis, it may be used as a new serological marker for non-invasive assessment of liver fibrosis.

Objective To investigate the differential expression of circular RNA ( circRNA ) in patients with chronic HBV infection of different stages.Methods Seven patients with chronic HBV infection admitted in Taizhou People's Hospital from October 2014 to October 2015 were enrolled, including 4 with chronic hepatitis B ( CHB ) and 3 chronic HBV carriers;3 healthy subjects served as controls. Peripheral blood mononuclear cells (PBMCs) were separated,and the expression of circRNA molecules in PBMCs were detected by new generation of circRNA microarray and validated by fluorescent quantitative PCR.The interaction sites between circRNA and miRNA were predicted with Arraystar miRNA target prediction software.Target genes regulated by the circRNA related to miRNA were analyzed by Gene oncology (Go) and Kyoto encyclopedia of genes and genomes (KEGG) analysis.SPSS 17.0 software was used for statistical analysis.Results Compared with the healthy controls , 137 circRNA molecules of differential expression were found in patients with chronic hepatitis B , of which 89 were up-regulated and 48 were down-regulated; while 444 circRNA molecules of differential expression , of which 130 were up-regulated (>5 fold in 34 ) and 314 down-regulated , were found in chronic HBV carriers.Compared with chronic HBV carriers , 1041 circRNA molecules of differential expression were found in CHB patients , including 663 up-regulated and 378 down-regulated (>5 fold in 54).There were many miRNA responsive elements which complementary with seed regions on miRNA in different circRNA molecules.Target gene analysis demonstrated that 533 target genes regulated by hsa_circ_0038646 were related to miRNAs , 249 target genes found in hsa_circ_0087354 were related to microRNAs.GO analysis showed that function of target genes regulated by hsa_circ_0038646 related to miRNA mainly enriched in activin binding.Function of target genes regulated by hsa_circ_0087354 related to miRNA mainly enriched in armadillo repeat domain binding.KEGG analysis showed that hsa_circ_0038646 molecules related to miRNA mainly involved in T cell receptor , estrogen receptor signaling pathway and so on.Hsa_circ_0087354 molecules related to miRNA mainly involved in adherens junction , MAPK signaling pathway and so on. Conclusion There are differential expressions of circRNA in patients at different clinical stages of chronic HBV infection , which might be involved in immune regulation of chronic HBV infection through the regulation of multiple target genes and signaling pathways.

Objective To investigate the effect of microRNA (miRNA )‐548ah targeting histone deacetylase‐4 (HDAC4) on the replication and expression of hepatitis B virus (HBV) .Methods HepG2 , 2 ,15 cells were transfected by mimics and inhibitors . The expressions of miRNA‐548ah and HDAC4 before and after transfection were detected by fluorescent quantitative polymerase chain reaction (PCR) . The expression of HDAC4 protein in HepG2 ,2 ,15 cells was detected by Western blotting .The target gene of miRNA‐548ah was analyzed by bioinformatics methods .3′UTR dual‐luciferase expression vector containing candidate HDAC4 target genes was built to test the luciferase activity .The levels of hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) in the supernatant of cultured HepG2 ,2 ,15 cells were detected by enzyme‐linked immunosorbent assay (ELISA) .The level of HBV DNA in the supernatant of HepG2 ,2 ,15 cells was detected by fluorescent quantitative PCR .The t‐test was used for comparison between two groups ,SNK‐q tests were used for multiple groups comparisons .Results The expressions of miRNA‐548ah in HepG2 ,2 ,15 and HepG2 cells were 5 .74 ± 0 .02 and 2 .96 ± 0 .40 , respectively (t= 11 .89 ,P< 0 .01) ,and the expressions of HDAC4 mRNA were 9 .38 ± 0 .39 and 18 .13 ±0 .34 ,respectively (t = 29 .39 , P < 0 .01) . The expression of miRNA‐548ah in HepG2 ,2 ,15 cells was inhibited by transfection of miRNA‐548ah inhibitors (1 .01 ± 0 .13 ,t= 15 .48 , P< 0 .01) .Compared with control group ,the levels of HBsAg ([6 .45 ± 0 .46 ] IU/mL vs [2 .60 ± 0 .20 ] IU/mL , t = 7 .48 , P <0 .01) ,HBeAg ([5 .49 ± 0 .27] NCU/mL vs [4 .15 ± 0 .34 ] NCU/mL , t = 3 .10 , P < 0 .05 ) and HBV DNA ([3 .93 ± 0 .06] lg copy/mL vs[2 .04 ± 0 .07] lg copy/mL ,t = 18 .89 , P< 0 .01) in the supernatant of cultured HepG2 ,2 ,15 cells significantly decreased in inhibitor group . The expression of HDAC4 in HepG2 ,2 ,15 cells significantly decreased after transfection of miRNA‐548ah mimics (2 .98 ± 0 .94) ,but significantly increased after transfection of miRNA‐548ah inhibitors (23 .77 ± 6 .74 ) , with statistical significance (F= 9 .34 , P< 0 .01) .The expression of HDAC4 protein was also significantly inhibited after transfection of miRNA‐548ah mimics (0 .53 ± 0 .14 vs 0 .23 ± 0 .02 , t = 3 .58 , P = 0 .02) .The activity of luciferase was significantly inhibited by transfection of miRNA‐548ah mimics (7 .62 ± 0 .45 vs 6 .65 ±0 .27 ,t = 3 .18 , P = 0 .03 ) .Conclusion miRNA‐548ah may promote the replication and expression of HBV through the regulation of target gene HDAC4 .

Objective To screen and analyze microRNA (miRNA) molecules associated with immune clearance in patients with chronic hepatitis B (CHB).Methods Twelve hospitalized CHB patients and 9 healthy individuals from Taizhou People's Hospital during June 2011 and August 2012 were enrolled.miRCURYTM chip was used to identify differential expression patterns of miRNA.The target genes of differentially expressed miRNA molecules were predicted by TargetScan,miRBase and miRanda databases,and their molecular pathways and functions were analyzed with bioinformatic methods.Results Compared with healthy individuals,52 differentially expressed miRNA molecules were identified in PBMCs of CHB patients,including 33 up-regulated and 19 down-regulated ones.With TargetScan,miRBase and miRanda databases,354 target genes were predicted in up-regulated miRNA molecules,and 1 935 target genes were predicted in down-regulated miRNA molecules.Gene ontology (GO) and Pathway analysis showed that several molecular pathways might be affected by up or down-regulated miRNA molecules.miRNA-mRNA network analysis showed that some target genes might be regulated by miRNA molecules like hsa-miR-520d-5p,hsa-miR-106a-Sp,hsa-miR-30a-5p and hsa-miR-29b-3p,and constituted a complex molecular network.Conclusion There are several miRNA molecules with abnormal expressions in CHB patients,which may be involved in immune clearance through the regulation of target genes and molecular pathways.

Objective To investigate the expression of microRNA-548ah (miR-548ah) and microRNA-4804 (miR-4804) in peripheral blood mononuclear cells (PBMCs) of patients with chronic hepatitis B virus infection and their clinical significance.Methods PBMCs were collected from 29 patients with chronic hepatitis B (CHB),30 hepatitis B virus carriers (HBVC),26 inactive HBsAg carriers (IASC) and 28 healthy controls in Taizhou People's Hospital during September 2012 and August 2013.Expressions of miR-548ah and miR-4804 in PBMCs were detected by fluorescence real-time quantitative RT-PCR (qRT-PCR).Receiver operating characteristic (ROC) curve was used to evaluate the expression of miR-548ah and miR-4804 in distinguishing immune tolerance phase and clearance phase of HBV infection.Pearson correlation analysis was performed to assess the correlations of miRNAs expression with clinical markers alanine aminotrans ferase (ALT) and HBV DNA loads.Results There were significant differences in expressions of miR-548ah and miR-4804 in PBMCs between CHB,HBVC,IASC groups and control group (F =28.16 and 83.17,P ＜ 0.01).Compared with control group,miR-548ah was up-regulated in CHB group(P ＜ 0.01),and down-regulated in HBVC and IASC groups (P ＜ 0.01) ; miR-4804 was up-regulated in CHB group (P ＜ 0.01),down-regulated in HBVC group (P ＜ 0.01),while there was no significant difference between IASC group and control group in miR-4804 expressions(P ＞ 0.05).The areas under ROC curve (AUCs) of miR-548ah and miR4804 in differentiation of immune tolerance and immune clearance were 0.966 and 0.997,and the sensitivities and specificities were 89.7％,96.6％ and 99.6％,100.0％,respectively.No significant correlation was found between the expression of miR-548ah,miR-4804 and ALT,HBV DNA loads (r=0.14,0.18,-0.20 and-0.19,P＞0.05).Conclusion miR-548ah and miR-4804 may be involved in the immunopathogenesis of CHB,and their expression levels in PBMCs are helpful in differentiation of immune tolerance and immune clearance in HBV infection.

Objective To explore the expression profiles and their clinical significance of microRNA (miRNA) molecules in different stages of chronic hepatitis B virus (HBV) infection.Methods The miRNA molecule expressions of 12 patients with chronic hepatitis B,12 chronic HBV carriers,12 inactive hepatitis B surface antigen (HBsAg) carriers,and 9 healthy controls were screened using miRNA microarray.The miRNA profiles were validated by the real time fluorescence quantitative polymerase chain reaction (PCR).The t-test was used for comparison between twogroups,whereas one-way ANOVA and SNK-q tests were used for multiple comparisons.Mann-Whitney and Kruskal Wallis H tests were used for comparison of two or more groups of data with skewed distribution.The receiver operation characteristic (ROC) curve was constructed to evaluate the diagnostic significance of miRNA molecules.Results Compared with the healthy controls,significant differences in the expression profiles of miRNA molecules were found in peripheral blood mononuclear cells of chronic HBV carriers (2 molecules up-regulated,and 18 down regulated) and chronic hepatitis B patients (33 molecules up-regulated and 19 down-regulated).No significant difference was found between inactive HBsAg carriers (2 molecules up regulated,and 3 down-regulated) and controls.The results of six miRNA molecules detected by real-time fluorescence quantitative PCR were basically consistent with the results detected by microarray.The area under ROC curve of the six miRNA molecules of hsa miR-4711-3p,hsa-miR-3191 5p,hsa-miR-5704-5p,hsa-miR 548ah-5p,hsa-miR-146a-5p and hsa-miR-29b-3p in distinguishing immune tolerance and clearance of chronic HBV infection were 0.994,0.984,0.967,1.000,1.000 and 0.996,respectively.Conclusions The different expression profiles of miRNA molecules could be used to distinguish the different stages of chronic HBV infection,and are closely related with the immune tolerance and activation in chronic HBV infection.

Objective miRNA-122 levels may correlate with liver cancer prognosis,and therefore understanding its expression is crucial for future treatment.This study investigates the effect of DNA methylation on the expression of liver specific miRNA-122 and its effects on proliferation and apoptosis of hepatocellular carcinoma cells.Methods Methylation sequencing detected the methylation of the miRNA-122 promoter region,and the level of miRNA-122 expression was measured by using real-time quantitative PCR.The proliferation and apoptosis of hepatocellular cell lines were detected by flow cytometry and CCK8.Results Compared with human primary hepatocytes [(21.9 ± 11.4)％],the level of miRNA-122 promoter methylation in Huh7,HepG2,and QSG-7701 cell lines were (87.6±9.3) ％,(89.0 ± 14.3)％,and (69.5 ±11.5)％,respectively.This represents a significant increase (P=0.000),especially in Huh7 and HepG2 cell lines.Compared with human primary hepatocytes (2.83× 104 ±3746),the levels of miR-122 expression in the above three cell lines were significantly decreased,especially in Huh7 and HepG2 cell lines (P=0.007).After treatment with 5-Aza-dc,the degree of methylation in Huh7 and HepG2 cell lines were significantly lower than that of the blank group (P=0.038,P=0.025),and the levels of miRNA-122 expression were significantly elevated (P=0.008,P=0.003).Also,compared with the control groups,the apoptosis of Huh7 cells and HepG2 cells were significantly increased (P=0.001,0.027).Conclusion The expression of miRNA-122 is regulated by DNA methylation and correlated with the apoptosis of liver cancer cells.Therfore,the methylation regulation of miRNA-122 expression might be involved in the occurrence and development of hepatocellular carcinoma.