Long-term antiviral therapy is associated with changes in the profile of transcriptionally active HBV integration in the livers of patients with CHB.

Hepatitis B virus (HBV) integration exists throughout the clinical course of chronic hepatitis B (CHB). This study investigated the effects of long-term antiviral therapy on the level and profiles of transcriptionally active HBV integration. Serial liver biopsies and paired blood samples were obtained from 16, 16, and 22 patients with CHB at baseline, 78, and 260 weeks of entecavir monotherapy or combined with pegylated interferon alfa, respectively. Serum HBV biomarkers were longitudinally assessed. RNA-seq and HIVID2 program was used to identify HBV-host chimeric RNAs transcribed from integrated DNA. The counts of HBV integration reads were positively related to both serum HBV DNA levels (r = 0.695, p = 0.004) and HBeAg titers (r = 0.724, p = 0.021) at baseline, but the positive correlation exited only to the serum HBsAg levels after 260 weeks of antiviral therapy (r = 0.662, p = 0.001). After 78 weeks of antiviral therapy, the levels of HBV integration expression decreased by 12.25 folds from baseline. The viral junction points were enriched at the S and HBx genes after the long-term antiviral therapy. HBs-FN1 became one of the main transcripts, with the mean proportion of HBs-FN1 in all integrated expression increased from 2.79% at baseline to 10.54% at Week 260 of antiviral treatment. Antiviral therapy may reduce but not eliminate the HBV integration events and integration expression. Certain integration events, such as HBs-FN1 can persist in long-term antiviral treatment.

An Immunocompetent Mongolian Gerbil Model for Hepatitis E Virus Genotype 1 Infection.

BACKGROUND & AIMS: Hepatitis E virus (HEV), primarily genotype 1 (HEV-1), causes approximately 20.1 million infections, 44,000 deaths, and 3000 stillbirths annually. Current evidence indicates that HEV-1 is only transmitted in humans. Here, we evaluated whether Mongolian gerbils can serve as animal models for HEV-1 infection. METHODS: Mongolian gerbils were used for HEV-1 and hepatitis E virus genotype 3 infection experiments. HEV infection parameters, including detection of HEV RNA and HEV antigen, liver function assessment, and histopathology, were evaluated. RESULTS: We adapted a clinical isolate of HEV-1 for Mongolian gerbils by serial passaging in feces of aged male gerbils. The gerbil-adapted strain obtained at passage 3 induced a robust, acute HEV infection, characterized by stable fecal virus shedding, elevated liver enzymes, histopathologic changes in the liver, and seroconversion to anti-HEV. An infectious complementary DNA clone of the adapted virus was generated. HEV-1-infected pregnant gerbils showed a high rate of maternal mortality and vertical transmission. HEV RNA or antigens were detected in the liver, kidney, intestine, placenta, testis, and fetus liver. Liver and placental transcriptomic analyses indicated activation of host immunity. Tacrolimus prolonged HEV-1 infection, whereas ribavirin cleared infection. The protective efficacy of a licensed HEV vaccine was validated using this model. CONCLUSIONS: HEV-1 efficiently infected Mongolian gerbils. This HEV-1 infection model will be valuable for investigating hepatitis E immunopathogenesis and evaluating vaccines and antivirals against HEV.

Multi-omics panoramic analysis of HBV integration, transcriptional regulation, and epigenetic modifications in PLC/PRF/5 cell line.

The clearance or transcriptional silencing of integrated HBV DNA is crucial for achieving a functional cure in patients with chronic hepatitis B and reducing the risk of hepatocellular carcinoma development. The PLC/PRF/5 cell line is commonly used as an in vitro model for studying HBV integration. In this study, we employed a range of multi-omics techniques to gain a panoramic understanding of the characteristics of HBV integration in PLC/PRF/5 cells and to reveal the transcriptional regulatory mechanisms of integrated HBV DNA. Transcriptome long-read sequencing (ONT) was conducted to analyze and characterize the transcriptional activity of different HBV DNA integration sites in PLC/PRF/5 cells. Additionally, we collected data related to epigenetic regulation, including whole-genome bisulfite sequencing (WGBS), histone chromatin immunoprecipitation sequencing (ChIP-seq), and assays for transposase-accessible chromatin using sequencing (ATAC-seq), to explore the potential mechanisms involved in the transcriptional regulation of integrated HBV DNA. Long-read RNA sequencing analysis revealed significant transcriptional differences at various integration sites in the PLC/PRF/5 cell line, with higher HBV DNA transcription levels at integration sites on chr11, chr13, and the chr13/chr5 fusion chromosome t (13:5). Combining long-read DNA and RNA sequencing results, we found that transcription of integrated HBV DNA generally starts downstream of the SP1, SP2, or XP promoters. ATAC-seq data confirmed that chromatin accessibility has limited influence on the transcription of integrated HBV DNA in the PLC/PRF/5 cell line. Analysis of WGBS data showed that the methylation intensity of integrated HBV DNA was highly negatively correlated with its transcription level (r = -0.8929, p = 0.0123). After AzaD treatment, the transcription level of integrated HBV DNA significantly increased, especially for the integration chr17, which had the highest level of methylation. Through ChIP-seq data, we observed the association between histone modification of H3K4me3 and H3K9me3 with the transcription of integrated HBV DNA. Our findings suggest that the SP1, SP2 and XP in integrated HBV DNA, methylation level of surrounding host chromosome, and histone modifications affect the transcription of integrated HBV DNA in PLC/PRF/5 cells. This provides important clues for future studies on the expression and regulatory mechanisms of integrated HBV.

Higher TP53BP2 expression is associated with HBsAg loss in peginterferon-α-treated patients with chronic hepatitis B.

BACKGROUND & AIMS: HBsAg loss is only observed in a small proportion of patients with chronic hepatitis B (CHB) who undergo interferon treatment. Investigating the host factors crucial for functional cure of CHB can aid in identifying individuals who would benefit from peginterferon-α (Peg-IFNα) therapy. METHODS: We conducted a genome-wide association study (GWAS) by enrolling 48 patients with CHB who achieved HBsAg loss after Peg-IFNα treatment and 47 patients who didn't. In the validation stage, we included 224 patients, of whom 90 had achieved HBsAg loss, to validate the identified significant single nucleotide polymorphisms. To verify the functional involvement of the candidate genes identified, we performed a series of in vitro and in vivo experiments. RESULTS: GWAS results indicated a significant association between the rs7519753 C allele and serum HBsAg loss in patients with CHB after Peg-IFNα treatment (p = 4.85 × 10-8, odds ratio = 14.47). This association was also observed in two independent validation cohorts. Expression quantitative trait locus analysis revealed higher hepatic TP53BP2 expression in individuals carrying the rs7519753 C allele (p = 2.90 × 10-6). RNA-sequencing of liver biopsies from patients with CHB after Peg-IFNα treatment revealed that hepatic TP53BP2 levels were significantly higher in the HBsAg loss group compared to the HBsAg persistence group (p = 0.035). In vitro and in vivo experiments demonstrated that loss of TP53BP2 decreased interferon-stimulated gene levels and the anti-HBV effect of IFN-α. Mechanistically, TP53BP2 was found to downregulate SOCS2, thereby facilitating JAK/STAT signaling. CONCLUSION: The rs7519753 C allele is associated with elevated hepatic TP53BP2 expression and an increased probability of serum HBsAg loss post-Peg-IFNα treatment in patients with CHB. TP53BP2 enhances the response of the hepatocyte to IFN-α by suppressing SOCS2 expression. IMPACT AND IMPLICATIONS: Chronic hepatitis B (CHB) remains a global public health issue. Although current antiviral therapies are more effective in halting disease progression, only a few patients achieve functional cure for hepatitis B with HBsAg loss, highlighting the urgent need for a cure for CHB. This study revealed that the rs7519753 C allele, which is associated with high expression of hepatic TP53BP2, significantly increases the likelihood of serum HBsAg loss in patients with CHB undergoing Peg-IFNα treatment. This finding not only provides a promising predictor for HBsAg loss but identifies a potential therapeutic target for Peg-IFNα treatment. We believe our results are of great interest to a wide range of stakeholders based on their potential clinical implications.

RNA binding protein TIAR modulates HBV replication by tipping the balance of pgRNA translation.

The pregenomic RNA (pgRNA) of hepatitis B virus (HBV) serves not only as a bicistronic message RNA to translate core protein (Cp) and DNA polymerase (Pol), but also as the template for reverse transcriptional replication of viral DNA upon packaging into nucleocapsid. Although it is well known that pgRNA translates much more Cp than Pol, the molecular mechanism underlying the regulation of Cp and Pol translation efficiency from pgRNA remains elusive. In this study, we systematically profiled HBV nucleocapsid- and pgRNA-associated cellular proteins by proteomic analysis and identified TIA-1-related protein (TIAR) as a novel cellular protein that binds pgRNA and promotes HBV DNA replication. Interestingly, loss- and gain-of-function genetic analyses showed that manipulation of TIAR expression did not alter the levels of HBV transcripts nor the secretion of HBsAg and HBeAg in human hepatoma cells supporting HBV replication. However, Ribo-seq and PRM-based mass spectrometry analyses demonstrated that TIAR increased the translation of Pol but decreased the translation of Cp from pgRNA. RNA immunoprecipitation (RIP) and pulldown assays further revealed that TIAR directly binds pgRNA at the 5' stem-loop (ε). Moreover, HBV replication or Cp expression induced the increased expression and redistribution of TIAR from the nucleus to the cytoplasm of hepatocytes. Our results thus imply that TIAR is a novel cellular factor that regulates HBV replication by binding to the 5' ε structure of pgRNA to tip the balance of Cp and Pol translation. Through induction of TIAR translocation from the nucleus to the cytoplasm, Cp indirectly regulates the Pol translation and balances Cp and Pol expression levels in infected hepatocytes to ensure efficient viral replication.

The A1762T/G1764A mutations enhance HBV replication by alternating viral transcriptome.

The A1762T/G1764A mutations, one of the most common mutations in the hepatitis B virus basal core promoter, are associated with the progression of chronic HBV infection. However, effects of these mutations on HBV replication remains controversial. This study aimed to systematically investigate the effect of the mutations on HBV replication and its underlying mechanisms. Using the prcccDNA/pCMV-Cre recombinant plasmid system, a prcccDNA-A1762T/G1764A mutant plasmid was constructed. Compared with wild-type HBV, A1762T/G1764A mutant HBV showed enhanced replication ability with higher secreted HBV DNA and RNA levels, while Southern and Northern blot indicated higher intracellular levels of relaxed circular DNA, single-stranded DNA, and 3.5 kb RNA. Meanwhile, the mutations increased expression of intracellular core protein and decreased the production of HBeAg and HBsAg. In vitro infection based on HepG2-NTCP cells and mice hydrodynamic injection experiment also proved that these mutations promote HBV replication. 5'-RACE assays showed that these mutations upregulated transcription of pregenomic RNA (pgRNA) while downregulating that of preC RNA, which was further confirmed by full-length transcriptome sequencing. Moreover, a proportion of sub-pgRNAs with the potential to express polymerase were also upregulated by these mutations. The ChIP-qPCR assay showed that A1762T/G1764A mutations created a functional HNF1α binding site in the BCP region, and its overexpression enhanced the effect of A1762T/G1764A mutations on HBV. Our findings revealed the mechanism and importance of A1762T/G1764A mutations as an indicator for management of CHB patients, and provided HNF1α as a new target for curing HBV-infected patients.

Integrated HBV DNA and cccDNA maintain transcriptional activity in intrahepatic HBsAg-positive patients with functional cure following PEG-IFN-based therapy.

BACKGROUND: Hepatitis B surface antigen (HBsAg) seroclearance marks regression of hepatitis B virus (HBV) infection. However, more than one-fifth of patients with functional cure following pegylated interferon-based therapy may experience HBsAg seroreversion. The mechanisms causing the HBV relapse remain unclear. AIM: To investigate the level and origin of HBV transcripts in patients with functional cure and their role in predicting relapse. METHODS: Liver tissue obtained from patients with functional cure, as well as uncured and treatment-naïve HBeAg-negative patients with chronic hepatitis B (CHB) were analysed for intrahepatic HBV markers. HBV capture and RNA sequencing were used to detect HBV integration and chimeric transcripts. RESULTS: Covalently closed circular DNA (cccDNA) levels and the proportion of HBsAg-positive hepatocytes in functionally cured patients were significantly lower than those in uncured and treatment-naïve HBeAg-negative patients. Integrated HBV DNA and chimeric transcripts declined in functionally cured patients compared to uncured patients. HBsAg-positive hepatocytes present in 25.5% of functionally cured patients, while intrahepatic HBV RNA remained in 72.2%. The levels of intrahepatic HBV RNA, integrated HBV DNA, and chimeric transcripts were higher in functionally cured patients with intrahepatic HBsAg than in those without. The residual intrahepatic HBsAg in functionally cured patients was mainly derived from transcriptionally active integrated HBV DNA; meanwhile, trace transcriptional activity of cccDNA could also remain. Two out of four functionally cured patients with intrahepatic HBsAg and trace active cccDNA experienced HBV relapse. CONCLUSION: Integrated HBV DNA and cccDNA maintain transcriptional activity and maybe involved in HBsAg seroreversion in intrahepatic HBsAg-positive patients with functional cure and linked to virological relapse.

AUF1 promotes hepatocellular carcinoma progression and chemo-resistance by post-transcriptionally upregulating alpha-fetoprotein expression.

BACKGROUND: The target genes of AU-rich Element RNA-binding Protein 1 (AUF1), which is an RNA binding protein, and its role in the progression of hepatocellular carcinoma (HCC) is still elusive. This study aims to investigate the biological function and the underlying target genes of AUF1 in HCC. METHODS: RNA sequencing data and the Liver Cancer Institute (LCI) database were used to screen candidate targets of AUF1. LCI database, TCGA database, and a retrospective HCC cohort were used to investigate the correlation between AUF1 and alpha-fetoprotein (AFP) and their prognostic values in HCC patients. Huh-7, HepG2, and HepAD38 cell lines were used to investigate the underlying mechanism of AUF1 regulating the AFP expression. Cell Counting Kit-8, colony formation, EdU incorporation, and flow cytometry assays were performed to detect the effect of AUF1-AFP axis on the progression and doxorubicin resistance of HCC cells. RESULTS: A combined analysis of the transcriptome data from Huh-7 cells after knockdown of AUF1 and gene expression data from LCI database revealed that AFP was the most significantly downregulated gene after AUF1 depletion. AUF1 expression was positively associated with AFP expression in HCC tissues and the high expression of both AUF1 and AFP were correlated with a worse prognosis in HCC patients of LCI and TCGA databases, as well as our retrospective cohort. Mechanistically, AUF1 bound to the 3' untranslation region (UTR) of AFP mRNA to enhance the mRNA stability of AFP, thereby upregulating AFP. Functional tests showed that AFP knockdown inhibited tumor growth and doxorubicin resistance of HCC cells induced by AUF1. CONCLUSIONS: AFP may be an important target gene of AUF1. AUF1 promoted HCC progression and doxorubicin resistance by upregulating AFP expression via increasing its mRNA stability.

LncCDCA3L inhibits cell proliferation via a novel RNA structure-based crosstalk with CDCA3 in hepatocellular carcinoma.

BACKGROUND & AIMS: The molecular mechanisms underlying hepatocellular carcinoma (HCC) remain poorly understood. In this study, we investigated cell division cycle-associated 3 (CDCA3) expression status and characterized a CDCA3-related long non-coding RNA (lncRNA) in HCC. METHODS: RT-qPCR and western blot were used to determine CDCA3 expression level in HCC clinical specimens. 5' and 3'-RACE, RNAscope, RNA pull-down, CRISPR/Cas9-based RNA immunoprecipitation (CRIP) and site-directed mutation experiments were used to characterize lncCDCA3L and investigate its function target. Chi-square test and Kaplan-Meier analysis were used to assess lncCDCA3L clinical significance. The effects of lncCDCA3L on HCC development were assessed by overexpression in vitro and in vivo. RESULTS: In this study, we found CDCA3 was a potential oncogenic factor in HCC and characterized the lncCDCA3L, which could inhibit CDCA3. LncCDCA3L is significantly downregulated in HCC and its expression level is associated with tumour size and can act as an independent risk factor affecting postoperative survival time in HCC patients. Mechanistically, lncCDCA3L can repress CDCA3 protein level and inhibit hepatocarcinogenesis by directly binding to CDCA3 mRNA at 1423-1455 region via a novel manner based on a hairpin structure motif. CONCLUSIONS: Our study collectively unveiled the molecular mechanisms of how lncCDCA3L repressed the tumourigenic properties of HCC cells and exhibited a tumour suppressor character in HCC in a CDCA3-dependent manner. The findings here support lncCDCA3L can be used as a candidate prognostic biomarker for HCC patients.

E2F1-mediated AUF1 upregulation promotes HCC development and enhances drug resistance via stabilization of AKR1B10.

The AU-rich binding factor 1 (AUF1) is one of the well known adenylate-uridylate-rich element (ARE)-specific RNA-binding proteins (ARE-BPs) for which dysregulation has been reported in various human cancers. However, the involvement of AUF1 in the initiation and progression of hepatocellular carcinoma (HCC) is still elusive. In this study, we aimed at exploring the clinical significance, function, and mechanism of the abnormal expression of AUF1 in HCC. Using a bioinformatics analysis of The Cancer Genome Atlas (TCGA) and Liver Cancer Institute (LCI) database, we identified that AUF1 was abnormally highly expressed in HCC tissues and that the high expression of AUF1 was correlated with poor prognosis in patients with HCC. We also confirmed the increased AUF1 expression and its prognostic value in our HBV-related HCC cohorts. AUF1 overexpression in hepatoma cells promoted cell proliferation and increased the resistance of hepatoma cells toward doxorubicin, whereas knockdown of AUF1 exerted the opposite effects. Mechanistically, we demonstrated that AKR1B10 was a critical target of AUF1 and was essential for sustaining the AUF1-induced proliferation and drug resistance of hepatoma cells. AUF1 increased AKR1B10 expression by binding to the 3'UTR region of AKR1B10 mRNA and stabilizing AKR1B10 mRNA. Additionally, we demonstrated that E2F1 enhanced AUF1 expression in HCC at the transcription level. Our study revealed a novel role of AUF1 in promoting the development and drug resistance of HCC via the post-transcriptional regulation of AKR1B10 expression. The E2F1/AUF1/AKR1B10 axis can serve as a potential therapeutic target in HCC.

Pacbio Sequencing of PLC/PRF/5 Cell Line and Clearance of HBV Integration Through CRISPR/Cas-9 System.

The integration of HBV DNA is one of the carcinogenic mechanisms of HBV. The clearance of HBV integration in hepatocyte is of great significance to cure chronic HBV infection and thereby prevent the occurrence of HBV-related hepatocellular carcinoma (HCC). However, the low throughput of traditional methods, such as Alu-PCR, results in low detecting sensitivity of HBV integration. Although the second-generation sequencing can obtain a large amount of sequencing data, but the sequencing fragments are extremely short, so it cannot fully explore the characteristics of HBV integration. In this study, we used the third-generation sequencing technology owning advantages both in sequencing length and in sequencing depth to analyze the HBV integration characteristics in PLC/PRF/5 cells comprehensively. A total of 4,142,311 cleaning reads was obtained, with an average length of 18,775.6 bp, of which 84 reads were fusion fragments of the HBV DNA and human genome. These 84 fragments located in seven chromosomes, including chr3, chr4, chr8, chr12, chr13, chr16, and chr17. We observed lots of DNA rearrangement both in the human genome and in HBV DNA fragments surrounding the HBV integration site, indicating the genome instability causing by HBV integration. By analyzing HBV integrated fragments of PLC/PRF/5 cells that can potentially express HBsAg, we selected three combinations of sgRNAs targeting the integrated fragments to knock them out with CRISPR/Cas9 system. We found that the sgRNA combinations could significantly decrease the level of HBsAg in the supernatant of PLC/PRF/5 cells, while accelerated cell proliferation. This study proved the effectiveness of third-generation sequencing to detect HBV integration, and provide a potential strategy to reach HBsAg clearance for chronic HBV infection patients, but the knock-out of HBV integration from human genome by CRISPR/Cas9 system may have a potential of carcinogenic risk.

NFATc3 inhibits hepatocarcinogenesis and HBV replication via positively regulating RIG-I-mediated interferon transcription.

Nuclear factor of activated T cells 3 (NFATc3) has been reported to upregulate type I interferons (IFNs) expression, and the abnormal expression and activation of NFATc3 were closely related to tumorigenesis. However, the potential function of NFATc3 in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) remains to be elucidated. In this study, we found that NFATc3 gene was frequently deleted and downregulated in HCC tumor tissues, and that the downregulation of NFATc3 was associated with poor prognosis of HCC patients. The gain- and loss-of-function experiments demonstrated that NFATc3 inhibited HCC cell proliferation and invasion, as well as HBV replication. Mechanistically, NFATc3 could bind to the promoters of IFNL1 and IFNB1 genes and prompt the production of IFNs and interferon-stimulated genes. Furthermore, retinoic acid-inducible gene-I (RIG-I) pathway activation increased NFATc3 expression and nuclear localization, and activated NFATc3 further enhanced RIG-I-mediated IFN responses. Collectively, our findings reveal a novel regulatory signaling cascade, the RIG-I/NFATc3/IFNs axis, which inhibits hepatocarcinogenesis and HBV replication by enhancing the immune response in hepatocytes, and this functional axis might potentially be exploited for therapeutic benefits in the clinical treatment of HBV-related HCC.

Sex-determining region Y box 4 (SOX4) suppresses Hepatitis B virus replication by inhibiting hepatocyte nuclear factor 4α expression.

Hepatitis B virus (HBV) infection is still a health care crisis in the world, and a considerable number of chronic hepatitis B patients die of end-stage liver diseases, including liver cirrhosis and hepatocellular carcinoma. A previous study has reported that sex-determining region Y box 4 (SOX4) promotes HBV replication by binding to the AACAAAG motif in the viral genome. However, such SOX4 binding site was not found in the genome of the majority of HBV genotype strains. Further, we found that SOX4 inhibited rather than promoted the replication of most HBV strains. In line with this, HBV replication was significantly enhanced when the endogenous SOX4 was knocked down. Moreover, we demonstrated that the SOX4-induced suppression of HBV replication was mainly mediated by hepatocyte nuclear factor 4α (HNF4α). Taken together, our findings suggest that SOX4 plays an important antiviral role by inhibiting HNF4α expression in most HBV strains.

Exploring the Diagnostic Potential of Serum Golgi Protein 73 for Hepatic Necroinflammation and Fibrosis in Chronic HCV Infection with Different Stages of Liver Injuries.

BACKGROUND AND AIM: Serum Golgi protein 73 (GP73) is a promising alternative biomarker of chronic liver diseases, but most data are from patients with HBV infection rather than HCV. MATERIALS AND METHODS: Two independent cohorts of chronic hepatitis C (CHC) patients from the 5th Medical Centre of the Chinese PLA General Hospital (n = 174) and Beijing Youan Hospital (n = 120) with different histories of HCV infection were enrolled. The correlations between serum GP73 and other biochemical indices, as well as its correlations with different stages of liver disease progression, were investigated. The receiver operating characteristic (ROC) curve was employed to evaluate the diagnostic potential of serum GP73 for liver necroinflammation and fibrosis, and comparisons of the diagnostic efficiency with traditional indices of hepatic liver injuries were further investigated. RESULTS: Levels of serum GP73 were found significantly elevated in patients with moderate to severe inflammatory grade (G ≥ 2) and/or with advanced fibrotic stages (F ≥ 3) in both cohorts (P < 0.05, respectively), as compared to those with a normal or mild liver lesion. Further ROC analysis demonstrated that serum GP73 was comparable to serum ALT and AST in diagnosing the liver necroinflammation grade at G ≥ 2, but its diagnostic values for advanced fibrosis (F ≥ 3) and cirrhosis (F = 4) were limited when compared to APRI and FIB-4, and FIB-4 exhibited the best performance. Notably, an obvious elevation of serum GP73 was observed after patients received PEG-IFN and ribavirin treatment. CONCLUSIONS: Serum GP73 is an important biomarker in evaluating and monitoring the disease progression including liver necroinflammation and fibrosis in patients with chronic HCV infection, but the value is limited for diagnosing advanced fibrosis and cirrhosis in comparison with APRI and FIB-4.

Notoamide-type alkaloid induced apoptosis and autophagy via a P38/JNK signaling pathway in hepatocellular carcinoma cells.

Bioassay-guided fractionation of a coral-associated fungus Aspergillus ochraceus LZDX-32-15 resulted in the isolation of eleven notoamide-type alkaloids, including four new congeners, namely notoamides W-Z (1-4). The structures of the new alkaloids were determined by extensive analyses of spectroscopic data (1D and 2D NMR, HRESIMS), while ECD data were used for the configurational assignment. Three alkaloids (6, 10, 11) exerted potent inhibition against a panel of hepatocellular carcinoma (HCC) cell lines with IC50 values ranging from 0.42 to 3.39 µM, that are comparable to the data for paclitaxel. Notoamide G (6) inhibited the viability of HepG2 and Huh-7 cells via both apoptosis and autophagy pathways. Notoamide G activated the expression of caspase-3, caspase-8, and caspase-9, in association with the degradation of the downstream gene PARP in a dose-dependent manner, suggesting that notoamide G induced apoptosis via a mitochondrial pathway and a dead receptor-mediated pathway. In addition, notoamide G increased the autophagic vacuole in both HepG2 and Huh-7 cells in a dose-dependent manner after 24 h through the significant upregulation of the key proteins Beclin1 and LC3B. Further investigation revealed that notoamide G promoted P38 and JNK phosphorylation, whereas the total protein of P-38 and JNK was slightly influenced. Accordingly, the antitumor proliferation of notoamide G in HCC cells was mechanistically mediated by apoptosis and autophagy through a P38/JNK signaling pathway, while notoamide G was considered as a potent lead for further development as an antitumor agent.

Compensatory upregulation of aldo-keto reductase 1B10 to protect hepatocytes against oxidative stress during hepatocarcinogenesis.

Aldo-keto reductase 1B10 (AKR1B10), a member of aldo-keto reductase superfamily, contributes to detoxification of xenobiotics and metabolization of physiological substrates. Although increased expression of AKR1B10 was found in hepatocellular carcinoma (HCC), the role of AKR1B10 in the development of HCC remains unclear. This study aims to illustrate the role of AKR1B10 in hepatocarcinogenesis based on its intrinsic oxidoreduction abilities. HCC cell lines with AKR1B10 overexpression or knockdown were treated with doxorubicin or hydrogen peroxide to determinate the influence of aberrant AKR1B10 expression on cells' response to oxidative stress. Using Akr1b8 (the ortholog of human AKR1B10) knockout mice, diethylnitrosamine (DEN) induced liver injury, chronic inflammation and hepatocarcinogenesis were explored. Clinically, the pattern of serum AKR1B10 relevant to disease progression was investigated in a patient cohort with chronic hepatitis B (n=30), liver cirrhosis (n=30) and HCC (n=40). AKR1B10 expression in HCC tissues was analyzed using both the TCGA database (n=371) and our collected HCC samples (n=67). AKR1B10 overexpression reduced hepatocyte injury while AKR1B10 knockdown augmented reactive oxygen species (ROS) accumulation and apoptotic cell death. Consistently, Akr1b8 deficiency in mice promoted DEN-induced hepatocyte damage and liver inflammation characterized by increased phospho-H2AX, serum alanine aminotransferase, interleukin-6 and tumor necrosis factor alpha level, myeloid cell infiltration and led to more severe hepatocarcinogenesis and metastasis compared with wild type mice due to significant alteration on detoxification and oxidoreduction. AKR1B10 was compensatory expressed and gradually upregulated in the process of liver disease progression in HCC and increased oxidative stress upregulated AKR1B10 through NRF2. Our results here suggested that through oxidoreduction and detoxification, AKR1B10 played an important role in protecting hepatocytes from damage induced by ROS. Deficiency of AKR1B10 might accelerate hepatotoxin and inflammation-associated hepatocarcinogenesis. AKR1B10 expression elevation in HCC could be a result of compensatory upregulation, rather than a driver of malignant transformation during the development of HCC.

Methylation of PCDH19 predicts poor prognosis of hepatocellular carcinoma.

OBJECTIVE: Several members of protocadherins (PCDHs) have been identified as tumor suppressor genes in human carcinogenesis, but little is known about PCDH19. The aim of the present study was to assess the expression and methylation of PCDH19 in hepatocellular carcinoma (HCC). METHODS: The RNA-seq data from The Cancer Genome Atlas Database were downloaded and used for analyzing PCDH19 expression in HCC patients and normal liver tissues. We collected 63 paired tumor and nontumor liver tissues from hepatitis B virus-related HCC patients. The expression of PCDH19 was detected by real-time quantitative RT-PCR assay. The methylation of PCDH19 gene was analyzed by DNA methylation-sensitive endonuclease digestion and the sequential quantitative PCR. The prognostic value of PCDH19 gene methylation was evaluated by Kaplan-Meier analyses. RESULTS: PCDH19 expression was downregulated in HCC tissues and seven HCC cell lines compared to nontumor tissues. PCHD19 promoter was frequently hypermethylated in three (SMMC7721, Hep3B and SNU387) of seven HCC cell lines and 5-aza-dC treatment could significantly increased the PCDH19 expression in these methylated cells. In addition, HCC tumor tissues exhibited significantly increased PCDH19 hypermethylation both in frequency (30.15% vs 9.52%, P = 0.003) and in intensity (P = 0.002) compared to that in nontumor tissues. Kaplan-Meier survival analysis revealed that PCDH19 hypermethylation was correlated with the poor overall survival of HCC patients. CONCLUSION: PCDH19 expression was downregulated in HCC, which was mediated at least in part by promoter hypermethylation. PCDH19 hypermethylation might present a potential prognostic marker in HCC patients.