HOXA-AS2 Epigenetically Inhibits HBV Transcription by Recruiting the MTA1-HDAC1/2 Deacetylase Complex to cccDNA Minichromosome.

Persistent transcription of HBV covalently closed circular DNA (cccDNA) is critical for chronic HBV infection. Silencing cccDNA transcription through epigenetic mechanisms offers an effective strategy to control HBV. Long non-coding RNAs (lncRNAs), as important epigenetic regulators, have an unclear role in cccDNA transcription regulation. In this study, lncRNA sequencing (lncRNA seq) is conducted on five pairs of HBV-positive and HBV-negative liver tissue. Through analysis, HOXA-AS2 (HOXA cluster antisense RNA 2) is identified as a significantly upregulated lncRNA in HBV-infected livers. Further experiments demonstrate that HBV DNA polymerase (DNA pol) induces HOXA-AS2 after establishing persistent high-level HBV replication. Functional studies reveal that HOXA-AS2 physically binds to cccDNA and significantly inhibits its transcription. Mechanistically, HOXA-AS2 recruits the MTA1-HDAC1/2 deacetylase complex to cccDNA minichromosome by physically interacting with metastasis associated 1 (MTA1) subunit, resulting in reduced acetylation of histone H3 at lysine 9 (H3K9ac) and lysine 27 (H3K27ac) associated with cccDNA and subsequently suppressing cccDNA transcription. Altogether, the study reveals a mechanism to self-limit HBV replication, wherein the upregulation of lncRNA HOXA-AS2, induced by HBV DNA pol, can epigenetically suppress cccDNA transcription.

Kinesin Family Member-18A (KIF18A) Promotes Cell Proliferation and Metastasis in Hepatocellular Carcinoma.

BACKGROUND & AIMS: Kinesin family member 18A (KIF18A) is notable for its aberrant expression across various cancer types and its pivotal role is driving cancer progression. In this study, we aim to investigate the intricate molecular mechanisms underlying the impact of KIF18A on the progression of HCC. METHODS: Western blotting assays, a quantitative real-time PCR and immunohistochemical analyses were performed to quantitatively assess KIF18A expression in HCC tissues. We then performed genetic manipulations within HCC cells by silencing endogenous KIF18A using short hairpin RNA (shRNA) and introducing exogenous plasmids to overexpress KIF18A. We monitored cell progression, analyzed cell cycle and cell apoptosis and assessed cell migration and invasion both in vitro and in vivo. Moreover, we conducted RNA-sequencing to explore KIF18A-related signaling pathways utilizing Reactome and KEGG enrichment methods and validated these critical mediators in these pathways. RESULTS: Analysis of the TCGA-LIHC database revealed pronounced overexpression of KIF18A in HCC tissues, the finding was subsequently confirmed through the analysis of clinical samples obtained from HCC patients. Notably, silencing KIF18A in cells led to an obvious inhibition of cell proliferation, migration and invasion in vitro. Furthermore, in subcutaneous and orthotopic xenograft models, suppression of KIF18A sgnificantly redudce tumor weight and the number of lung metastatic nodules. Mechanistically, KIF18A appears to facilitate cell proliferation by upregulating MAD2 and CDK1/CyclinB1 expression levels, with the activation of SMAD2/3 signaling contributing to KIF18A-driven metastasis. CONCLUSION: Our study elucidates the molecular mechanism by which KIF18A mediates proliferation and metastasis in HCC cells, offering new insights into potential therapeutic targets.

RNA-binding protein RPS7 promotes hepatocellular carcinoma progression via LOXL2-dependent activation of ITGB1/FAK/SRC signaling.

BACKGROUND: Metastasis is one of the leading cause contributes to treatment failure and poor prognosis of hepatocellular carcinoma (HCC) patients. The underlying mechanism of HCC metastasis remains to be determined. Although several RNA binding proteins (RBPs) have been found to participate in tumorigenesis and progression of liver cancer, the role of RBPs in HCC patients with extrahepatic metastases is poorly understood. METHODS: By performing RNA-seq of primary HCC tissues (including HCC with extrahepatic metastasis and those did not develop metastasis), we identified a set of HCC metastasis-associated RBPs candidates. Among which, ribosomal protein S7 (RPS7) was found to be remarkably increased in HCC tissues and be strongly related to HCC poor survival. Overexpression or CRISPR-Cas9-mediated knockout were applied to investigate the role of RPS7 on the metastasis-associated phenotypes of HCC cells. RNA sequencing, RIP, RNA-pull down, dual luciferase reporter assay, nascent RNA capture assay, and RNA decay and so on, were applied to reveal the underlying mechanism of RPS7 induced HCC metastasis. RESULTS: Gain- and loss- of function analyses revealed that RPS7 promoted HCC cells adhesion, migration and invasion capabilities, as well as lung metastasis. Mechanistically, we uncovered that lysyl oxidase-like 2 (LOXL2) was a critical downstream target of RPS7. RPS7 could stabilize LOXL2 mRNA by binding to AUUUA motifs in the 3155-3375 region of the 3'UTR of LOXL2 mRNA, thus increased LOXL2 expression via elevating LOXL2 mRNA abundance. Further research revealed that LOXL2 could accelerate focal adhesion formation through maintaining the protein stability of ITGB1 and activating ITGB1-mediated FAK/SRC signaling pathway, and thereby contribute to the pro-metastasis effect of RPS7. CONCLUSIONS: Taken together, our data reveal a novel function of RPS7 in HCC metastasis, also reveal the critical roles of the RPS7/LOXL2/ITGB1 axis in HCC metastasis and shed new light on the exploration of molecular drugs against HCC.

ZNF148 inhibits HBV replication by downregulating RXRα transcription.

BACKGROUND: Progressive hepatitis B virus (HBV) infection can result in cirrhosis, hepatocellular cancer, and chronic hepatitis. While antiviral drugs that are now on the market are efficient in controlling HBV infection, finding a functional cure is still quite difficult. Identifying host factors involved in regulating the HBV life cycle will contribute to the development of new antiviral strategies. Zinc finger proteins have a significant function in HBV replication, according to earlier studies. Zinc finger protein 148 (ZNF148), a zinc finger transcription factor, regulates the expression of various genes by specifically binding to GC-rich sequences within promoter regions. The function of ZNF148 in HBV replication was investigated in this study. METHODS: HepG2-Na+/taurocholate cotransporting polypeptide (HepG2-NTCP) cells and Huh7 cells were used to evaluate the function of ZNF148 in vitro. Northern blotting and real-time PCR were used to quantify the amount of viral RNA. Southern blotting and real-time PCR were used to quantify the amount of viral DNA. Viral protein levels were elevated, according to the Western blot results. Dual-luciferase reporter assays were used to examine the transcriptional activity of viral promoters. ZNF148's impact on HBV in vivo was investigated using an established rcccDNA mouse model. RESULTS: ZNF148 overexpression significantly decreased the levels of HBV RNAs and HBV core DNA in HBV-infected HepG2-NTCP cells and Huh7 cells expressing prcccDNA. Silencing ZNF148 exhibited the opposite effects in both cell lines. Furthermore, ZNF148 inhibited the activity of HBV ENII/Cp and the transcriptional activity of cccDNA. Mechanistic studies revealed that ZNF148 attenuated retinoid X receptor alpha (RXRα) expression by binding to the RXRα promoter sequence. RXRα binding site mutation or RXRα overexpression abolished the suppressive effect of ZNF148 on HBV replication. The inhibitory effect of ZNF148 was also observed in the rcccDNA mouse model. CONCLUSIONS: ZNF148 inhibited HBV replication by downregulating RXRα transcription. Our findings reveal that ZNF148 may be a new target for anti-HBV strategies.

Inhibiting cholesterol de novo synthesis promotes hepatocellular carcinoma progression by upregulating prostaglandin E synthase 2-mediated arachidonic acid metabolism under high fatty acid conditions.

Inhibition of cholesterol de novo synthesis (DNS) by statins has controversial effects on the treatment of hepatocellular carcinoma (HCC). High fatty acid conditions have been reported to limit the effect of statins on metabolism diseases. Whether high fatty acid conditions interfere with the effect of statins on HCC remains unclear. Here, we reported that inhibiting cholesterol DNS with atorvastatin promoted the oncogenic capabilities of diethylnitrosamine (DEN) in mice fed high fatty acid diets (HFD). The combined analysis of metabolomics and transcriptomics revealed that arachidonic acid (AA) metabolism was the most significant changed pathway between mice with and without atorvastatin treatment. In vitro, in the presence of AA precursor linoleic acid (LA), atorvastatin promoted the proliferation and migration ability of HCC cell lines. However, in the absence of LA, these phenomena disappeared. TCGA and tissue microarray examination revealed that prostaglandin e synthase 2 (PTGES2), a key enzyme in AA metabolism, was associated with the poor outcome of HCC patients. Overexpression of PTGES2 promoted the proliferation and migration of HCC cell lines, and knockdown of PTGES2 inhibited the proliferation and migration of cells. Additionally, atorvastatin upregulated PTGES2 expression by enhancing Sterol-regulatory element binding protein 2 (SREBP2)-mediated transcription. Knockdown of PTGES2 reversed the proliferation and migration ability enhanced by atorvastatin. Overall, our study reveals that a high fatty acid background is one of the possible conditions limiting the application of statins in HCC, under which statins promote the progression of HCC by enhancing SREBP2-mediated PTGES2 transcription.

Gambogic acid inhibits HBx-mediated hepatitis B virus replication by targeting the DTX1-Notch signaling pathway.

BACKGROUND & AIMS: Current antiviral drugs, including nucleoside analogs and interferon, fail to eliminate the HBV covalently closed circular DNA (cccDNA), which serves as a transcript template in infected hepatocytes. Silencing the HBV X protein, which plays a crucial role in cccDNA transcription, is a promising approach to inhibit HBV replication. Therefore, the identification of novel compounds that can inhibit HBx-mediated cccDNA transcription is critical. METHODS: Initially, a compound library consisting of 715 monomers derived from traditional Chinese medicines known for their liver-protecting properties was established. Then, MTT assays were used to determine the cytotoxicity of each compound. The effect of candidates on Flag-HBx expression was examined by real-time PCR and western blotting in Flag-HBx transfected HepG2-NTCP cells. Ultimately, the antiviral effect of gambogic acid (GA) on HBV was observed in HBV-infected HepG2-NTCP cells. Mechanistically, the functional role of DTX1 in GA-induced HBV inhibition was examined using RNA-seq. Finally, the antiviral effect of GA was estimated in vivo. RESULTS: Gambogic acid (GA), a natural bioactive compound with a myriad of biological activities, markedly reduced Flag-HBx expression. Potent and dose-dependent reductions in extracellular HBV RNAs, HBV DNA, HBsAg, HBeAg and HBc protein were discovered three days after GA treatment in HBV-infected cells, accompanied by the absence of significant cytotoxicity. Furthermore, our research revealed that GA exhibited a dose-dependent inhibition of HBx expression, which is a pleiotropic protein required for HBV infection in vivo. We explored the mechanisms underlying GA-mediated inhibition of HBV and confirmed that this inhibition is accomplished by upregulating the expression of the DTX1 gene and boosting the Notch signaling pathway. Finally, the inhibitory effect of GA on HBV replication was tested in vivo using a mouse model of hepatitis B virus recombinant cccDNA. CONCLUSIONS: Herein, we discovered GA, which is a natural bioactive compound that targets HBx to inhibit hepatitis B virus replication by activating the DTX1-Notch signaling pathway.

Fault Diagnosis of Planetary Gearbox Based on Dynamic Simulation and Partial Transfer Learning.

To address the problem of insufficient real-world data on planetary gearboxes, which makes it difficult to diagnose faults using deep learning methods, it is possible to obtain sufficient simulation fault data through dynamic simulation models and then reduce the difference between simulation data and real data using transfer learning methods, thereby applying diagnostic knowledge from simulation data to real planetary gearboxes. However, the label space of real data may be a subset of the label space of simulation data. In this case, existing transfer learning methods are susceptible to interference from outlier label spaces in simulation data, resulting in mismatching. To address this issue, this paper introduces multiple domain classifiers and a weighted learning scheme on the basis of existing domain adversarial transfer learning methods to evaluate the transferability of simulation data and adaptively measure their contribution to label predictor and domain classifiers, filter the interference of unrelated categories of simulation data, and achieve accurate matching of real data. Finally, partial transfer experiments are conducted to verify the effectiveness of the proposed method, and the experimental results show that the diagnostic accuracy of this method is higher than existing transfer learning methods.

Research on the Influence of Geometric Structure Parameters of Eddy Current Testing Probe on Sensor Resolution.

To study the influence of the geometric structure of the probe coil on the electromagnetic characteristics of the eddy current probe in the process of eddy current testing, based on the principle of eddy current testing, different probe coil models were established using finite element software. These geometric structure parameters include the difference between the inner and outer radius, thickness, and equivalent radius. The magnetic field distribution around the probe is simulated and analyzed under different parameters, and the detection performance of the probe is judged in combination with the change rate of the magnetic field around the probe coil. The simulation results show that at a closer position, increasing the difference between the inner and outer radii, reducing the thickness, and reducing the equivalent radius are beneficial to improve the resolution of the probe coil. At a far position, reducing the difference between the inner and outer radii, increasing the thickness, and reducing the equivalent radius are beneficial to improve the resolution of the probe coil. At the same time, the accuracy of the simulation data is verified by comparing the theoretical values with the simulated values under different conditions. Therefore, the obtained conclusions can provide a reference and basis for the optimal design of the probe structure.

Sphondin efficiently blocks HBsAg production and cccDNA transcription through promoting HBx degradation.

Hepatitis B surface antigen (HBsAg) loss and seroconversion, which is considered as functional cure of chronic Hepatitis B virus (HBV) infection, is rarely achieved even after long-term antiviral treatments. Therefore, new antiviral strategies interfering with other HBV replication steps are required, especially those that could efficiently inhibit HBsAg production. Here, we identified novel anti-HBV compounds that could potently block HBsAg expression from cccDNA by screening a natural compound library derived from Chinese traditional medical plants by a novel screening strategy. The combination of ELISA assay detecting the HBsAg and real-time PCR detecting HBV RNAs as indicator for cccDNA transcriptional activity were used. The antiviral activity of a candidate compound and underlying mechanism were evaluated in HBV-infected cells and a humanized liver mouse model. Herein, we selected a highly effective low-cytotoxic compound sphondin, which could effectively inhibit both intracellular HBsAg production and HBV RNAs levels. Moreover, we found that sphondin markedly inhibited cccDNA transcriptional activity without affecting cccDNA level. Mechanistic study found sphondin preferentially bound to HBx protein by residue Arg72, which led to increased 26S proteasome-mediated degradation of HBx. Sphondin treatment significantly reduced the recruitment of HBx to cccDNA, which subsequently led to inhibition of cccDNA transcription and HBsAg expression. The absence of HBx or R72A mutation potently abrogated the antiviral effect induced by sphondin in HBV-infected cells. Collectively, sphondin may be considered as a novel and natural antiviral agent directly targeting HBx protein, which effectively inhibited cccDNA transcription and HBsAg expression.

Genome-wide association study of SARS-CoV-2 infection in Chinese population.

Coronavirus disease 2019 (COVID-19) is a global public health concern. The purpose of this study was to investigate the association between genetic variants and SARS-CoV-2 infection and the COVID-19 severity in Chinese population. A total of 256 individuals including 87 symptomatic patients (tested positive for SARS-CoV-2), 84 asymptomatic cases, and 85 close contacts of confirmed patients (tested negative for SARS-CoV-2) were recruited from February 2020 to May 2020. We carried out the whole exome genome sequencing between the individuals and conducted a genetic association study for SARS-CoV-2 infection and the COVID-19 severity. In total, we analyzed more than 100,000 single-nucleotide polymorphisms. The genome-wide association study suggested potential correlation between genetic variability in POLR2A, ANKRD27, MAN1A2, and ERAP1 genes and SARS-CoV-2 infection susceptibility. The most significant gene locus associated with SARS-CoV-2 infection was located in POLR2A (p = 5.71 × 10-6). Furthermore, genetic variants in PCNX2, CD200R1L, ZMAT3, PLCL2, NEIL3, and LINC00700 genes (p < 1 × 10-5) were closely associated with the COVID-19 severity in Chinese population. Our study confirmed that new genetic variant loci had significant association with SARS-CoV-2 infection and the COVID-19 severity in Chinese population, which provided new clues for the studies on the susceptibility of SARS-CoV-2 infection and the COVID-19 severity. These findings may give a better understanding on the molecular pathogenesis of COVID-19 and genetic basis of heterogeneous susceptibility, with potential impact on new therapeutic options.

Rapamycin inhibits hepatitis B virus covalently closed circular DNA transcription by enhancing the ubiquitination of HBx.

Hepatitis B virus (HBV) infection is still a serious public health problem worldwide. Antiviral therapies such as interferon and nucleos(t)ide analogs efficiently control HBV replication, but they cannot eradicate chronic hepatitis B (CHB) because of their incapacity to eliminate endocellular covalently closed circular DNA (cccDNA). Thus, there is a necessity to develop new strategies for targeting cccDNA. As cccDNA is difficult to clear, transcriptional silencing of cccDNA is a possible effective strategy. HBx plays a vitally important role in maintaining the transcriptional activity of cccDNA and it could be a target for blocking the transcription of cccDNA. To screen new drugs that may contribute to antiviral therapy, the ability of 2,000 small-molecule compounds to inhibit HBx was examined by the HiBiT lytic detection system. We found that the macrolide compound rapamycin, which is clinically used to prevent acute rejection after organ transplantation, could significantly reduce HBx protein expression. Mechanistic studies demonstrated that rapamycin decreased the stability of the HBx protein by promoting its degradation via the ubiquitin-proteasome system. Moreover, rapamycin inhibited HBV RNA, HBV DNA, and cccDNA transcription levels in HBV-infected cells. In addition, HBx deficiency abrogated the inhibition of cccDNA transcription induced by rapamycin. Similar results were also confirmed in a recombinant cccDNA mouse model. In summary, we report a new small-molecule, rapamycin, which targets HBx to block HBV cccDNA transcription and inhibit HBV replication. This approach can identify new strategies to cure CHB.

HBx Mediated Increase of DDX17 Contributes to HBV-Related Hepatocellular Carcinoma Tumorigenesis.

HBV is strongly associated with HCC development and DEAD-box RNA helicase 17 (DDX17) is a very important member of the DEAD box family that plays key roles in HCC development by promoting cancer metastasis. However, the important role of DDX17 in the pathogenesis of HBV-related HCC remains unclear. In this study, we investigated the role of DDX17 in the replication of HBV and the development of HBV-associated HCC. Based on data from the GEO database and HBV-infected cells, we found that DDX17 was upregulated by the HBV viral protein X (HBx). Mechanistically, increased DDX17 expression promoted HBV replication and transcription by upregulating ZWINT. Further study showed that DDX17 could promote HBx-mediated HCC metastasis. Finally, the promotive effect of DDX17 on HBV and HBV-related HCC was confirmed in vivo. In summary, the results revealed the novel role of DDX17 in the replication of HBV and the metastasis of HBV-associated HCC.

SIRT2 Promotes HBV Transcription and Replication by Targeting Transcription Factor p53 to Increase the Activities of HBV Enhancers and Promoters.

Chronic hepatitis B (CHB) virus infection is one of the leading causes of cirrhosis and liver cancer. Although the major drugs against CHB including nucleos(t)ide analogs and PEG-interferon can effectively control human hepatitis B virus (HBV) infection, complete cure of HBV infection is quite rare. Targeting host factors involved in the viral life cycle contributes to developing innovative therapeutic strategies to improve HBV clearance. In this study, we found that the mRNA and protein levels of SIRT2, a class III histone deacetylase, were significantly upregulated in CHB patients, and that SIRT2 protein level was positively correlated with HBV viral load, HBsAg/HBeAg levels, HBcrAg, and ALT/AST levels. Functional analysis confirmed that ectopic SIRT2 overexpression markedly increased total HBV RNAs, 3.5-kb RNA and HBV core DNA in HBV-infected HepG2-Na+/taurocholate cotransporting polypeptide cells and primary human hepatocytes. In contrast, SIRT2 silencing inhibited HBV transcription and replication. In addition, we found a positive correlation between SIRT2 expression and HBV RNAs synthesis as well as HBV covalently closed circular DNA transcriptional activity. A mechanistic study suggested that SIRT2 enhances the activities of HBV enhancer I/HBx promoter (EnI/Xp) and enhancer II/HBc promoter (EnII/Cp) by targeting the transcription factor p53. The levels of HBV EnI/Xp and EnII/Cp-bound p53 were modulated by SIRT2. Both the mutation of p53 binding sites in EnI/Xp and EnII/Cp as well as overexpression of p53 abolished the effect of SIRT2 on HBV transcription and replication. In conclusion, our study reveals that, in terms of host factors, a SIRT2-targeted program might be a more effective therapeutic strategy for HBV infection.

Pimobendan Inhibits HBV Transcription and Replication by Suppressing HBV Promoters Activity.

Current anti-HBV therapeutic strategy relies on interferon and nucleos(t)ide-type drugs with the limitation of functional cure, inducing hepatitis B surface antigen (HBsAg) loss in very few patients. Notably, the level of HBsAg has been established as an accurate indicator to evaluate the drug efficacy and predict the disease prognosis, thus exploring a novel drug targeting HBsAg will be of great significance. Herein, by screening 978 compounds from an FDA-approved drug library and determining the inhibitory function of each drug on HBsAg level in HepG2.2.15 cells supernatant, we identified that pimobendan (Pim) has a powerful antiviral activity with relatively low cytotoxicity. The inhibitory effect of Pim on HBsAg as well as other HBV markers was validated in HBV-infected cell models and HBV-transgenic mice. Mechanistically, real-time PCR and dual-luciferase reporter assay were applied to identify the partial correlation of transcription factor CAAT enhancer-binding protein α (C/EBPα) with the cccDNA transcription regulated by Pim. This indicates Pim is an inhibitor of HBV transcription through suppressing HBV promoters to reduce HBV RNAs levels and HBsAg production. In conclusion, Pim was identified to be a transcription inhibitor of cccDNA, thereby inhibiting HBsAg and other HBV replicative intermediates both in vitro and in vivo. This report may provide a promising lead for the development of new anti-HBV agent.

Glutamate dehydrogenase 1 mediated glutaminolysis sustains HCC cells survival under glucose deprivation.

Besides aerobic glycolysis, glutaminolysis has also become a hot spot in the field of tumor research because of its important role in regulating cell proliferation, apoptosis, and migration and invasion. Meanwhile, it is generally believed that tumor cells could sustain its proliferation and survival according to a so-called metabolic flexibility. How the metabolic flexibility of HCC cells behaves has not yet been fully elucidated. In this study, we validated the glutamine addiction of HCC cells, and identified that the glutaminolysis pathway of HCC cells altered in response to different glucose conditions. That is, glutamate transaminases GOT1 pathway played a dominant role in regulating cell growth when glucose was sufficient, yet deaminase GDH1 mediated metabolic pathway became dominant when glucose was limited, for the reason that GDH1 could drive the TCA cycle in response to glucose deprivation. Additionally, we further uncovered an negative relationship between GDH1 and GOT1 in low-glucose HCC tissues. Together, our study provided a new insight into the metabolic flexibility of glutaminolysis related enzymes in HCC, and highlighted the crucial role of GDH1 on HCC cells proliferation and survival in glucose starvation.

DDX17-regulated alternative splicing that produced an oncogenic isoform of PXN-AS1 to promote HCC metastasis.

BACKGROUND AND AIMS: The mechanism underlying HCC metastasis remains unclear, many oncogenes are known to regulate this process. However, the role of alternative splicing (AS) in pro-metastatic HCC is poorly understood. APPROACH AND RESULTS: By performing RNA sequencing on nine pairs of primary HCC tissues with extrahepatic metastasis (EHMH) and nine pairs of metastasis-free HCC (MFH) tissues, we depicted the AS landscape in HCC and found a higher frequency of AS events in EHMH compared with MFH. Moreover, 28 differentially expressed splicing regulators were identified in EHMH compared with MFH. Among these, DEAD-box RNA helicase 17 (DDX17) was significantly up-regulated in EHMH and was strongly associated with patient outcome. Functional studies indicated that DDX17 knockout inhibited the degradation of the extracellular matrix, and diminished the invasive ability of HCC cells. A significant reduction in lung metastasis induced by DDX17 deficiency was also demonstrated in a diethylnitrosamine-induced DDX17HKO mouse model. Mechanistically, high DDX17 induced intron 3 retention of PXN-AS1 and produced a transcript (termed PXN-AS1-IR3). The transcript PXN-AS1-IR3 acted as an important promoter of HCC metastasis by inducing MYC transcription activation via recruiting the complex of testis expressed 10 and p300 to the MYC enhancer region, which led to transcriptional activation of several metastasis-associated downstream genes. Finally, the PXN-AS1-IR3 level was significantly higher in serum and HCC tissues with extrahepatic metastasis. CONCLUSIONS: DDX17 and PXN-AS1-IR3 act as important metastatic promoters by modulating MYC signaling, suggesting that DDX17 and PXN-AS1-IR3 may be potential prognostic markers for metastatic HCC.

SIRT7 restricts HBV transcription and replication through catalyzing desuccinylation of histone H3 associated with cccDNA minichromosome.

Chronic hepatitis B virus (HBV) infection is a significant public health burden worldwide. HBV covalently closed circular DNA (cccDNA) organized as a minichromosome in nucleus is responsible for viral persistence and is the key obstacle for a cure of chronic hepatitis B (CHB). Recent studies suggest cccDNA transcription is epigenetically regulated by histone modifications, especially histone acetylation and methylation. In the present study, we identified transcriptionally active histone succinylation (H3K122succ) as a new histone modification on cccDNA minichromosome by using cccDNA ChIP-Seq approach. Silent mating type information regulation 2 homolog 7 (SIRT7), as an NAD+-dependent histone desuccinylase, could bind to cccDNA through interaction with HBV core protein where it catalyzed histone 3 lysine 122 (H3K122) desuccinylation. Moreover, SIRT7 acts cooperatively with histone methyltransferase, suppressor of variegation 3-9 homolog 1 (SUV39H1) and SET domain containing 2 (SETD2) to induce silencing of HBV transcription through modulation of chromatin structure. Our data improved the understanding of histone modifications of the cccDNA minichromosome, thus transcriptional silencing of cccDNA may represent a novel antiviral strategy for the prevention or treatment of HBV infection.

KAT2A Promotes Hepatitis B Virus Transcription and Replication Through Epigenetic Regulation of cccDNA Minichromosome.

Hepatitis B virus (HBV) infection remains a major health problem worldwide. Sufficient maintenance of the HBV covalently closed circular DNA (cccDNA), which serves as a template for HBV transcription, is responsible for the failure of antiviral therapies. While accumulating evidence suggests that cccDNA transcription is regulated by epigenetic machinery, particularly the acetylation and methylation of cccDNA-bound histone 3 (H3) and histone 4 (H4), the potential contributions of histone succinylation and related host factors remain obscured. Here, by screening a series of succinyltransferases and desuccinylases, we identified KAT2A as an important host factor of HBV transcription and replication. By using HBV-infected cells and mouse models with HBV infection, KAT2A was found to affect the transcriptional activity of cccDNA but did not affect cccDNA production. Mechanism studies showed that KAT2A is mainly located in the nucleus and could bind to cccDNA through interaction with HBV core protein (HBc). Moreover, we confirmed histone H3K79 succinylation (H3K79succ) as a histone modification on cccDNA minichromosome by using the cccDNA ChIP-Seq approach. Importantly, KAT2A silencing specifically reduced the level of cccDNA-bound succinylated H3K79. In conclusion, KAT2A promotes HBV transcription and replication through epigenetic machinery, and our findings may provide new insight into the treatment of HBV infection.

Dicoumarol, an NQO1 inhibitor, blocks cccDNA transcription by promoting degradation of HBx.

BACKGROUND & AIMS: Current antiviral therapies help keep HBV under control, but they are not curative, as they are unable to eliminate the intracellular viral replication intermediate termed covalently closed circular DNA (cccDNA). Therefore, there remains an urgent need to develop strategies to cure CHB. Functional silencing of cccDNA is a crucial curative strategy that may be achieved by targeting the viral protein HBx. METHODS: We screened 2,000 small-molecule compounds for their ability to inhibit HiBiT-tagged HBx (HiBiT-HBx) expression by using a HiBiT lytic detection system. The antiviral activity of a candidate compound and underlying mechanism of its effect on cccDNA transcription were evaluated in HBV-infected cells and a humanised liver mouse model. RESULTS: Dicoumarol, an inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), significantly reduced HBx expression. Moreover, dicoumarol showed potent antiviral activity against HBV RNAs, HBV DNA, HBsAg and HBc protein in HBV-infected cells and a humanised liver mouse model. Mechanistic studies demonstrated that endogenous NQO1 binds to and protects HBx protein from 20S proteasome-mediated degradation. NQO1 knockdown or dicoumarol treatment significantly reduced the recruitment of HBx to cccDNA and inhibited the transcriptional activity of cccDNA, which was associated with the establishment of a repressive chromatin state. The absence of HBx markedly blocked the antiviral effect induced by NQO1 knockdown or dicoumarol treatment in HBV-infected cells. CONCLUSIONS: Herein, we report on a novel small molecule that targets HBx to combat chronic HBV infection; we also reveal that NQO1 has a role in HBV replication through the regulation of HBx protein stability. LAY SUMMARY: Current antiviral therapies for hepatitis B are not curative because of their inability to eliminate covalently closed circular DNA (cccDNA), which persists in the nuclei of infected cells. HBV X (HBx) protein has an important role in regulating cccDNA transcription. Thus, targeting HBx to silence cccDNA transcription could be an important curative strategy. We identified that the small molecule dicoumarol could block cccDNA transcription by promoting HBx degradation; this is a promising therapeutic strategy for the treatment of chronic hepatitis B.