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ANXA4 restricts HBV replication by inhibiting autophagic degradation of MCM2 in chronic hepatitis B

BMC Medicine volume 22, Article number: 521 (2024) Cite this article

Abstract

Background

Hepatitis B virus (HBV) is an enveloped DNA virus that causes chronic hepatitis B (CHB) infection. Annexin, a Ca2+-activated protein, is widely expressed in various organs and tissues and has potential utility in disease diagnosis and treatment. However, the relationship between the annexin family and CHB remains unclear.

Methods

Clinical samples from hepatitis patients and donors or healthy individuals were collected. Transcriptome sequencing in CHB liver tissues and HBV-infected cells were performed. HepG2.2.15 cells with the full-length HBV genome and HBV-infected HepG2-NTCP cell models were established. HBV-infected mouse model was constructed and adeno-associated virus was utilized.

Results

ANXA4 expression was elevated during CHB infection. ANXA4 knockdown promoted HBV replication and aggravated liver injury, while ANXA4 overexpression alleviated that. Mechanistically, autophagy pathway was activated by ANXA4 deficiency, promoting autophagic degradation of minichromosome maintenance complex component 2 (MCM2). MCM2 inhibition activated HBV replication, while MCM2 overexpression attenuated ANXA4 deficiency-induced HBV replication and liver injury. Clinically, the expression of hepatitis B viral protein was negatively correlated with the ANXA4 levels, and CHB patients with high ANXA4 levels (> 8 ng/ml) showed higher sensitivity to interferon therapy.

Conclusions

ANXA4 functions as a protective factor during HBV infection. ANXA4 expression is elevated under HBV attack to restrict HBV replication by inhibiting autophagic degradation of MCM2, thereby alleviating liver injury and suppressing the CHB infection process. ANXA4 also enhances the sensitivity of CHB patients to interferon therapy. Therefore, ANXA4 is expected to be a new target for CHB treatment and prognostic evaluation.

Graphical Abstract

Highlights

• ANXA4 expression is elevated during CHB infection in both patients and mice.

• ANXA4 knockdown promotes HBV replication and transcription and aggravates liver injury.

• Autophagy is activated when ANXA4 deficiency, promoting autophagic degradation of MCM2.

• Inhibition of MCM2 expression activates HBV replication directly, while MCM2 overexpression attenuates ANXA4 deficiency-induced HBV replication and liver injury.

• The expression of hepatitis B viral protein is negatively correlated with the ANXA4 levels, and ANXA4 enhances the sensitivity of CHB patients to interferon therapy.

Peer Review reports

Background

Hepatitis B virus (HBV) is a small, enveloped DNA virus that causes acute and chronic liver diseases. Despite the availability of effective preventive vaccines in recent decades, chronic hepatitis B (CHB) infection remains a major global public health problem, with an estimated 887,000 deaths per year due to HBV infection [1,2,3,4,5]. Elucidating the pathogenesis of HBV is thus important for the cure of CHB.

HBV pathogenicity is related to the bidirectional interaction between the virus and the host. Microorganisms rely on annexins on the cell membrane to bind to and enter host cells. Annexin A4 (ANXA4), a member of the annexin family, is widely expressed in various organs and tissues and involved in endocytosis and exocytosis, cell proliferation and death process, playing an important role in liver diseases [6,7,8,9]. Previous proteomic sequencing on patients with different degrees of HBV-related fibrosis showed that the severity of fibrosis was related to the ANXA4 expression level [10]. In addition, ANXA4 was significantly increased in the saliva of patients infected with coronavirus disease 2019, indicating the correlation between ANXA4 and the response to viral infection and suggesting that ANXA4 may be related to the protective response of antiviral defense [11]. However, the mechanism of ANXA4 in HBV replication and transcription remains unclear.

Autophagy is a catabolic process for maintaining cellular homeostasis [12]. Upon mild liver injury, autophagy was activated to protect against cell death; however, autophagy was inhibited upon severe liver injury [13], which was important for the HBV replication cycle and non-replicative infection. HBV infection can also affect autophagy, and autophagy was involved in HBV amplification [14, 15]. Autophagy was simultaneously thought to be defensive responses to virus infection [16, 17]. For example, autophagy inhibition can reduce the response to herpes simplex virus infection [18] and Kaposi’s sarcoma herpesvirus infection [19]. Therefore, the role of autophagy in HBV replication and whether autophagy is involved in ANXA4-mediated HBV transcription also need to be clarified.

In this study, we found that ANXA4 is upregulated during HBV infection and the upregulated ANXA4 can inhibit the HBV replication and transcription processes. Mechanistically, ANXA4 reduces autophagic degradation of minichromosome maintenance complex component 2 (MCM2), and the accumulated MCM2 in turn inhibits HBV replication to alleviate liver injury and suppress the progression of CHB infection. Clinically, hepatitis B viral protein expression was negatively correlated with the ANXA4 levels, and CHB patients with high ANXA4 levels (> 8 ng/ml) showed higher sensitivity to interferon therapy. We thus suggest that ANXA4 plays a protective role in HBV infection, and ANXA4 is expected to be a new target for the treatment and prognostic evaluation of CHB infection.

Methods

Ethics statements

The protocol for the study with clinical samples was approved by the Medical Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University. The experimental animals were inspected and approved by the Institutional Animal Care and Use Committee of South China Agricultural University.

Clinical specimens

Clinical sample collection was approved by the Medical Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University (approval number: 20210228701). Serum samples were obtained from hepatitis patients and healthy volunteers. Liver tissue samples were obtained from patients by liver puncture, and normal liver tissue specimens were obtained by donation after cardiac death (DCD) during liver transplantation. Written informed consent was obtained from all patients and volunteers before inclusion in the study.

Establishment and treatment of HBV-infected mice

The experimental mice (C57BL/6j, 8-week-old, male) were purchased from Guangdong Scarstar Biotechnology Co., Ltd. (Guangzhou, China) and housed under pathogen-free conditions. The mouse model of HBV infection was established by tail vein injection of recombinant adeno-associated HBV (AAV-HBV). To inhibit ANXA4, recombinant adeno-associated virus containing shANXA4 (AAV-shANXA4) was further injected. The dose of AAV-HBV and AAV-shANXA4 was 1E + 11 genome copies (GC) (dissolved in 200 µL of 1 × phosphate-buffered saline (PBS)). The day of the first tail vein injection was recorded as day 0. Peripheral blood was collected once weekly for 8 weeks by rotating capillary blood vessels (with an inner diameter of approximately 1.0 mm) along the inner canthal orbit into the posterior retroorbital venous plexus. Furthermore, MCM2 recombinant protein was administered by tail vein injection to evaluate the effect of MCM2 on HBV replication (0.5 µg/mouse/time, 3 times weekly for 6 weeks). At the observation endpoint, mouse livers were harvested for subsequent experiments.

Cell culture

HepG2, Hep3B, HepG2.2.15, and PLC/PRF/5 cells were obtained from Guangdong Key Laboratory of Liver Disease Research, the Third Affiliated Hospital of Sun Yat-sen University (Guangzhou, China). HepG2-NTCP cells were obtained from the School of Basic Medical Sciences of Wuhan University (Wuhan, China). HepAD38 cells were acquired from the School of Basic Medical Sciences, Peking University Health Science Center (Beijing, China). HepG2, Hep3B, HepG2.2.15, and PLC/PRF/5 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (C11995500BT, Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (FBS, FSP500, ExCell Bio, China). HepG2-NTCP and HepAD38 cells were maintained in DMEM supplemented with 10% FBS and 400 μg of G418 per ml (11811031, Thermo Fisher Scientific, USA). All cells were cultured in a humidified incubator at 37 °C with 5% CO2.

Virus production and cell infection

HBV particles were collected and concentrated from the supernatant of HepAD38 cells. Cell supernatants were collected, and polyethylene glycol (PEG) 8000 was added at a final concentration of 5%. After thorough mixing by inversion and incubation with gentle shaking overnight at 4 °C, the mixture was centrifuged at 1000 × g for 60 min at 4 °C to collect HBV particles. The pellet was redissolved in serum-free Opti-MEM to 1% of the original supernatant sample volume. During infection, cells were seeded in collagen-coated 24-well plates, and HBV particles were diluted in DMEM supplemented with 10% FBS, 2% dimethyl sulfoxide (DMSO), 4% PEG8000, and 1% penicillin–streptomycin (P/S). Infection was performed at a multiplicity of infection (MOI) of 300. Twenty-four hours post-infection, the cells were washed three times with PBS for further experiments.

RNA sequencing

Total RNA was extracted from liver tissues or cell samples. Sequencing libraries were prepared according to the manufacturer’s instruction with steps including purifying mRNA and fragmenting it into small pieces, synthesizing first-strand cDNA, synthesizing second-strand cDNA, performing end repair by incubation with A-Tailing Mix, and ligating RNA Index Adapters. The products of the above steps were purified by AMPure XP Beads and then dissolved in EB solution. The product was validated on an Agilent Technologies 2100 bioanalyzer for quality control. The final library was amplified with phi29 to make DNA nanoballs (DNBs), which contained more than 300 copies of one molecule. DNBs were loaded into a patterned nanoarray, and single-end 50-base reads were generated on the MGIseq2000 platform (BGI-Shenzhen, China).

DNA and RNA extraction and quantitative RT-PCR (RT-qPCR)

Total DNA was extracted with a QIAamp DNA Mini Kit following the manufacturer’s protocol (51304, QIAGEN, Germany). Total RNA was purified by TRIzol reagent (15596018, Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s instruction. A quantity of 2 µg of RNA was used to synthesize cDNA, and 2 µL of cDNA was analyzed with the Light Cycler 480 instrument (Roche, Basel, Switzerland). The expression of every target gene was normalized to that of the β-actin gene from the same sample, and the values are presented as fold changes relative to the matched control values. Primer sequences are provided in Additional file 1.

Quantification of intracellular and intrahepatic covalently closed circular DNA (cccDNA)

HBV cccDNA was quantified by fluorescent probe quantitative PCR assays (SUPBIO Biotechnology, Guangzhou, China). Firstly, HBV cccDNA was purified from cells and liver tissues using QIAamp DNA Mini Kits (Qiagen, Hilden, Germany) following the manufacturer’s instruction and then the DNA was denatured at 85 °C for 5 min. Plasmid-safe ATP-dependent DNase was used to digest HBV rcDNA, replicative dsDNA, and ssDNA [20]. HBV cccDNA was quantified by qPCR with a primer pair and a probe targeting the gap region of the HBV genome. qPCR reactions were performed with an Applied Biosystems 7500 real-time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The quantification range of cccDNA was 10 to 1 × 106 copies/µL.

Quantification of intracellular and intrahepatic pre-genomic RNA (pgRNA)

pgRNA levels were measured at Guangzhou SupBio Biotechnology and Science Co., Ltd Lab by PCR fluorescent probing with the HBV Pre-genomic RNA Detection Kit (SUPBIO, #SUPI-0208, Guangzhou, China). HBV pgRNA in cells and mouse liver tissues was isolated and treated with DNase I, and then reverse-transcribed with a commercial kit according to the manufacturer’s instruction. The linear range was 5 × 101 to 1 × 108 copies/ml, with a LLoD of 15 copies/ml.

Measure of the serum HBsAg and HBeAg expression

Serum HBsAg and HBeAg levels were quantified by Roche immunochemistry analyzers (cobas e801 analyzers, Mannheim, Germany) according to the manufacturer’s instruction. The samples were diluted 10- to 40-fold before testing, and the measured values were multiplied by the dilution factor.

Determination of the supernatant HBsAg and HBeAg levels

Cell culture supernatant HBsAg (KHHBSAG, KHB, Shanghai, China) and HBeAg (KHHBEAG, KHB, Shanghai, China) levels were detected by enzyme-linked immunosorbent assay (ELISA).

Immunohistochemical (IHC) analysis

Formalin-fixed and paraffin-embedded liver tissue was sectioned into slices. After deparaffinization, the specimens were incubated with the indicated primary antibody at 4 °C overnight and with the secondary antibody for 30 min at room temperature. Diaminobenzidine (DAB; G1212-200 T, Servicebio, Wuhan, China) staining was used to visualize immunoreactivity. Nuclei were counterstained using hematoxylin (G1004-100ML, Servicebio, Wuhan, China). The antibodies used for immunohistochemical analysis are listed in Additional file 2.

Immunofluorescence (IF) analysis

Cells were fixed with 4% paraformaldehyde for 20 min. After washing with PBS, the cells were permeabilized in 0.3% Triton X-100 for 20 min. Then, the cells were blocked in PBS containing 2% bovine serum albumin (BSA) for 1 h. Cells were incubated with anti-ANXA4, anti-HBsAg, or anti-HBcAg antibodies at 4 °C overnight. After washing with PBS three times, the cells were incubated with Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies for 1 h at room temperature. 4′,6-Diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. For tissue section staining, the steps after dewaxing and antigen retrieval were similar to those described above. Images were acquired using a confocal laser scanning microscope (Leica DMi8, Leica, Wetzlar, Germany). The antibodies used for immunofluorescence analysis are listed in Additional file 2.

Western blotting (WB) and immunoprecipitation (IP)

Total tissue or cellular protein was purified, and the corresponding secondary antibodies were used to detect primary antibody/antigen complexes. Quantitative densitometric analysis of the indicated protein band was performed with normalization to β-actin [21]. Information regarding the antibodies used for western blot analysis is provided in Additional file 2. For co-immunoprecipitation (Co-IP), cells were lysed in Pierce™ IP Lysis Buffer with protease inhibitor, precleared with protein A magnetic beads (Bio-ray, 1,614,013) overnight at 4 °C, and then incubated with anti-ANXA4, anti-LC3B, anti-p62, or anti-MCM2 antibodies or with IgG for 2 h at room temperature. Finally, the magnetic beads were washed, boiled for 10 min at 70 °C, and harvested for western blot analysis. IgG was used as a control.

Statistical analysis

The results are presented as the mean ± SEM values. Statistical analyses were performed using Student’s t-test or the Mann–Whitney U test and one-way ANOVA by SPSS 22.0 and Prism 8 software (GraphPad Software Inc. La Jolla, CA, USA). Differences were considered significant when p < 0.05. NS, not significant (p > 0.05); *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

Results

ANXA4 expression is elevated during CHB infection

We firstly collected liver tissues for transcriptome sequencing and found that the differentially expressed genes (DEGs) in liver tissues of patients with CHB compared with normal liver tissues obtained from donors, and the enriched pathways were associated with inflammation (Additional file 3: Fig. S1A–B). We further investigated the role of the ANXA family in HBV infection, and the volcano plot and heatmap showed that ANXA4 was significantly upregulated in the CHB group (Additional file 3: Fig. S1C). Furthermore, we generated HBV-infected HepG2-NTCP cells, and the RNA-seq results also suggested that ANXA4 was markedly elevated during HBV infection among the ANXA family members (Fig. 1A–C). In subsequent verification analyses, HBV RNA and HBV DNA were detected in HBV-infected HepG2-NTCP cells (Fig. 1D). In addition, ANXA4 expression increased after HBV infection (Fig. 1E–F), consistent with the sequencing results. To test the specific correlation between ANXA4 and HBV, we found that the level of ANXA4 in hepatocytes with HBV genome integration was higher than that in hepatocytes without HBV genome integration (Fig. 1G–H). In hepatitis patients with different etiologies, the ANXA4 level in the CHB group was significantly higher than that in patients with hepatitis of other etiologies (Fig. 1I–K). In conclusion, ANXA4 is elevated during CHB infection and may be involved in HBV replication and liver injury.

Fig. 1
figure 1

ANXA4 expression is elevated during CHB infection. A–B Gene expression heatmap and volcano plot of ANXA family in HBV-infected HepG2-NTCP cells and non-HBV-infected HepG2-NTCP cells. C ANXA4 FPKM values in the sequencing group. D HBV RNA and HBV DNA levels in the HBV-infected and control groups. E ANXA4 mRNA levels in the HBV-infected and control groups. F ANXA4 protein expression in the infected group versus the control group. G ANXA4 mRNA levels in HBV-related cells. H ANXA4 protein expression levels in HBV-related cells. I The levels of ANXA4 mRNA expression in liver tissues of healthy individuals and hepatitis patients with different etiologies were determined by RT-qPCR. J ANXA4 protein expression in CHB and healthy liver tissues. K Immunohistochemistry results of ANXA4 expression in liver tissues (bar = 100 µm). The values are presented as the means ± SEMs. * p < 0.05, ** p < 0.01, *** p < 0.001

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ANXA4 inhibits HBV replication and viral protein expression

As a stress response under HBV infection, ANXA4 expression was upregulated. We then explored the effects of ANXA4 on HBV replication. HepG2.2.15 cells with the full-length HBV genome and HBV-infected HepG2-NTCP cell models were established. Firstly, we used lentivirus-mediated shRNA transduction to silence ANXA4 expression (Fig. 2A–B) and found that ANXA4 knockdown increased both the intracellular and supernatant levels of cccDNA, pgRNA, HBV DNA, HBV total RNA, HBsAg, and HBeAg levels (Fig. 2D–K). Immunofluorescence (IF) analysis also showed the increased expression of viral protein HBsAg and HBcAg after ANXA4 inhibition (Fig. 2L). On the other hand, the increased HBsAg and HBeAg levels in the supernatant may be explained by the enhanced cell death, not only due to the activated HBV replication. Accordingly, TUNEL assay was further applied to estimate the effect of ANXA4 on cell death. We found downregulation of ANXA4 in HepG2.2.15 cells and HepG2-NTCP cells infected with HBV did not increase cell death (Fig. 2C). Thus, the increase in HBsAg and HBeAg levels in the supernatant could be mainly explained by the enhanced HBV replication when ANXA4 was downregulated. In contrast, ANXA4 overexpression inhibited HBV replication and viral protein expression in both HepG2.2.15 cells and HBV-infected HepG2-NTCP cells (Additional file 3: Fig. S2A–K). Therefore, these findings reveal that ANXA4 inhibits HBV replication and viral protein expression.

Fig. 2
figure 2

ANXA4 inhibits HBV replication and viral protein expression. A, B ANXA4 knockdown was validated at the mRNA and protein level. C TUNEL staining in HepG2.2.15 and HepG2-NTCP + HBV cells with shCont or shANXA4 transfection (bar = 50 µm). D, H Intracellular ccDNA and pgRNA levels. E, I Intracellular and supernatant levels of HBV DNA. F, J Total HBV RNA levels. G, K Levels of HBsAg and HBeAg in the cell supernatant. L HBsAg and HBcAg immunofluorescence in HepG2.2.15 cells and HBV-infected HepG2-NTCP cells transfected with shCont or shANXA4 (bar = 50 µm). The values are presented as the means ± SEMs. * p < 0.05, ** p < 0.01, *** p < 0.001

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ANXA4 attenuates HBV replication and liver injury in mice and contributes to CHB clinical therapy

Next, a mouse model of HBV infection was established (Additional file 3: Fig. S3A). After 8 weeks of AAV-HBV infection, the HBV DNA, HBV RNA, HBsAg, and HBeAg levels in liver tissue were significantly increased (Additional file 3: Fig. S3B–C, E–F). Moreover, ANXA4 expression was higher in HBV-infected mice than that in non-HBV-infected mice (Additional file 3: Fig. S3D–F). Further, ANXA4 was specifically knocked down in the liver using adeno-associated viruses (Fig. 3A–B). The HBV DNA and HBV RNA levels in liver tissues were increased by ANXA4 knockdown (Fig. 3C), and the expression of the viral protein HBcAg and HBsAg was higher in the ANXA4-deficient group (Fig. 3D). In addition, the serum albumin (ALB) level was obviously decreased in the ANXA4-deficient group compared with the control group after HBV infection, and the ALT, AST, and TBA levels were also slightly increased after ANXA4 knockdown (Fig. 3E). Continuous analysis of HBV indexes in peripheral blood showed that the expression of HBeAg in the ANXA4-deficient group was significantly higher than that in the control group from 2 to 8 weeks and that the levels of HBV DNA and HBsAg showed the same trend at 3 to 8 weeks (Fig. 3F). The results of immunohistochemical and immunofluorescence also confirmed that ANXA4 depletion promoted HBsAg and HBcAg expression in HBV-infected mice, suggesting enhanced HBV replication (Fig. 3G, Additional file 3: Fig. S4A–D). Overall, our data confirm that specific knockdown of ANXA4 promotes HBV replication and transcription to aggravate liver injury.

Fig. 3
figure 3

ANXA4 attenuates HBV replication and liver injury in mice. A Flow chart of the experimental setup. B ANXA4 mRNA expression in liver tissues from HBV-infected mice treated with AAV-shCont or AAV-shANXA4. C Levels of HBV RNA and HBV DNA in mouse liver tissues. D The expression levels of HBsAg, HBcAg, and ANXA4 in the above groups were measured by WB. E Measurement of serum biochemical indexes in the above mice. F Serum HBV genomic DNA was quantified by real-time PCR. Serum HBeAg and HBsAg were quantified by a chemiluminescent microparticle immunoassay. G ANXA4, HBcAg, and HBsAg levels were determined by IF analysis (bar = 50 µm). The values are presented as the means ± SEMs. * p < 0.05, ** p < 0.01, *** p < 0.001

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To highlight the clinical translational proficiency of ANXA4 in CHB patients, we further evaluated the serum ANXA4 levels and found that ANXA4 was also significantly increased in the serum of CHB patients (Additional file 3: Fig. S5A) and the ANXA4 level in serum was positively correlated with its expression in the liver (Additional file 3: Fig. S5B). Basing that, we wonder the clinical relevance between ANXA4 and CHB. Firstly, additional information of the clinical characteristics (including age, sex, male gender, female gender, AST, ALT, TB, WBC, platelet count, NEUT, hemoglobin, FBG, AFP, TSH, FT3, FT4) was summarized to exclude other parameters that might also correlated with the ANXA4 level and cure rate, and results showed no statistically significant baseline between the cured and uncured groups (Additional file 4). Then, the serum samples from CHB patients revealed that HBsAg expression was negatively correlated with ANXA4 levels in these patients who were initially diagnosed with CHB. That is, CHB patients with high ANXA4 expression showed lower HBsAg levels (Additional file 3: Fig. S5C), suggesting that ANXA4 may inhibit HBV protein expression. Then, the patients were averagely divided into three groups based on their initial ANXA4 levels and for subsequent interferon therapy. We tracked their responses to interferon therapy and found the cure rate in the ANXA4 high-expression group (ANXA4 > 8 ng/ml, cure rate = 75%) was significantly higher than that in the ANXA4 middle-expression group (ANXA4 = 4 ~ 8 ng/ml, cure rate = 37.5%) and ANXA4 low-expression group (ANXA4 = 0 ~ 4 ng/ml, cure rate = 22.5%) (Additional file 3: Fig. S5D). By interferon therapy for 12 or 24 weeks, CHB patients in the ANXA4 high-expression group showed a greater decline rate of HBsAg compared with the other two groups (Additional file 3: Fig. S5E). In addition, clinical cured CHB patients with higher initial ANXA4 levels showed a shorter clinical cure time (Additional file 3: Fig. S5F). These clinical data further underline the correlation between HBV protein expression and ANXA4 level. ANXA4 may be involved in inhibiting HBV replication and enhancing the sensitivity of CHB patients to interferon therapy.

The autophagy pathway is activated when ANXA4 is inhibited

To explore the mechanism by which ANXA4 affects HBV replication, HBV-infected HepG2-NTCP cells with and without ANXA4 knockdown were subjected to transcriptome sequencing (Fig. 4A–C). Enrichment analysis suggested that autophagy-related pathways were activated (Fig. 4D). Transmission electron microscopy and confocal microscopy further showed that ANXA4 inhibition increased autophagosome formation in HepG2.2.15 cells and HBV-infected HepG2-NTCP cells (Fig. 4E), while ANXA4 overexpression inhibited that (Additional file 3: Fig. S6A). Measurement of mRNA (Atg5, Atg7, Atg12) and protein (LC3B, p62) levels suggested that autophagy was activated by ANXA4 knockdown (Fig. 4F–G). Moreover, HBcAg and HBsAg expression increased under ANXA4 inhibition (Fig. 4G–H). Additionally, ANXA4 overexpression inhibited autophagic process in normal and HBV-infected hepatocytes, and also suppressed HBV replication in HepG2.2.15 cells and HBV-infected HepG2-NTCP cells (Additional file 3: Fig. S6B–E). These results suggest that ANXA4 knockdown activates autophagy pathway during HBV infection, a phenomenon that might be associated with HBV replication.

Fig. 4
figure 4

The autophagy pathway is activated when ANXA4 is inhibited. A PCA of sequencing samples. B Volcano plot of DEGs. C Histogram of DEGs. A total of 1372 DEGs were upregulated and 778 were downregulated in HBV-infected HepG2-NTCP cells transduced with shANXA4 compared to those transduced with shCont. D Top 15 enriched KEGG pathways. E Transmission electron microscopy (bar = 2 µm) and fluorescence microscopy (bar = 10 µm) indicated that ANXA4 downregulation increased autophagosome formation. The red arrows indicate autophagosomes. F The mRNA levels of autophagy markers were measured by RT-qPCR. G ANXA4, LC3B, p62, and HBcAg expression was analyzed by WB. H HBsAg and HBcAg immunofluorescence in HepG2 2.2.15 cells and HBV-infected HepG2-NTCP cells treated with si-NC or si-ANXA4 (bar = 50 µm). The values are presented as the means ± SEMs. * p < 0.05, ** p < 0.01, *** p < 0.001

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Inhibition of ANXA4 promotes HBV replication via activating autophagy

The RNA-seq results indicated that the DNA replication pathway showed the largest enrichment ratio (0.92) after ANXA4 inhibition (Fig. 5A). HBV is a DNA virus and genes related to the DNA replication pathway play important roles in HBV replication and transcription initiation. Combining with the potential relationship of autophagy and HBV replication, we firstly explored the effects of autophagy inhibition on HBV replication. WB, RT-qPCR, and IF experiments proved that autophagy inhibitor 3-methyladenine (3-MA) and chloroquine (CQ) could directly suppress HBV replication in HepG2.2.15 and HepG2-NTCP + HBV cells (Additional file 3: Fig. S7A–K). Next, we explored whether ANXA4 inhibition enhances HBV replication via autophagy activation. 3-MA and CQ treatment both reversed the high expression of HBV proteins resulting from ANXA4 downregulation in HepG2.2.15 cells and HBV-infected HepG2-NTCP cells (Fig. 5B). Inhibition of autophagy attenuated the production of intracellular HBV DNA and HBV RNA and the secretion of cccDNA, pgRNA, HBV DNA, HBsAg, and HBeAg resulting from ANXA4 downregulation (Fig. 5C–J). Immunofluorescence staining supported the above conclusions (Fig. 5K). Thus, inhibition of ANXA4-mediated HBV replication activity is achieved by autophagy activation, and autophagy is a crucial regulator of the ANXA4-mediated HBV replication pathway.

Fig. 5
figure 5

Inhibition of ANXA4 promotes HBV replication via activating autophagy. A Enrichment ratios of KEGG pathways in HBV-infected HepG2-NTCP cells. B p62 and HBcAg levels in shCont- and shANXA4-transduced HepG2 2.2.15 cells and HBV-infected HepG2-NTCP cells treated with CQ or 3-MA. C cccDNA and pgRNA levels in HepG2.2.15 cells with above treatments. D HBV DNA and HBV RNA were detected in HepG2.2.15 cells. E–F HBV DNA, HBsAg, and HBeAg secreted by HepG2.2.15 cells were detected. G HBV DNA and HBV RNA levels in HBV-infected HepG2-NTCP cells were measured by RT-qPCR. H cccDNA and pgRNA levels in HBV-infected HepG2-NTCP cells with above treatments. I–J HBV DNA and HBV RNA in HBV-infected HepG2-NTCP cells were detected. K HBsAg and HBcAg expression was analyzed by immunofluorescence staining (bar = 50 µm). The values are presented as the means ± SEMs. * p < 0.05, ** p < 0.01, *** p < 0.001

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ANXA4 silencing enhances HBV replication by promoting autophagic degradation of MCM2

Minichromosome maintenance (MCM) complex components and gene stability regulatory factors in the DNA replication pathway were downregulated after ANXA4 knockdown, with MCM2 showing the most significant downregulation (Fig. 6A–C). In HepG2.2.15 cells and HBV-infected HepG2-NTCP cells, ANXA4 knockdown downregulated MCM2 expression (Fig. 6D–E and G–H), while MCM2 expression was not downregulated by ANXA4 knockdown in HepG2 cells and HepG2-NTCP cells without HBV infection (Additional file 3: Fig. S8A–D). Meanwhile, ANXA4 overexpression promoted MCM2 expression (Additional file 3: Fig. S8E–H). In addition, Earle’s balanced salt solution (EBSS) and rapamycin (RAPA) were used to determine the effects of autophagy activation on the degradation of MCM2. The results showed that autophagy induced by EBSS or rapamycin did not affect MCM2 degradation in HepG2 cells and HepG2-NTCP cells without HBV infection, while autophagy activation promoted the degradation of MCM2 in HepG2.2.15 cells and HepG2-NTCP + HBV cells with HBV infection (Additional file 3: Fig. S9A–B). Further, autophagy inhibition by 3-MA or CQ reversed the downregulation of MCM2 mediated by ANXA4 knockdown (Fig. 6F, I). The co-immunoprecipitation assays further demonstrated that ANXA4 bound to p62 and LC3B but could not interact with MCM2. The interaction between p62 and LC3B was enhanced after ANXA4 inhibition to promote autophagy (Fig. 6J). Moreover, MCM2 bound to p62, and inhibition of ANXA4 increased this interaction to promote autophagic degradation of MCM2 and reduce its expression (Fig. 6J). Simultaneously, as for the subcellular localization of MCM2 and p62, MCM2 was mostly nuclei localization and p62 was mostly cytoplasm localization in the normal condition. However, during CHB infection, the expression of MCM2 in the nucleus increased significantly and partially transferred to the cytoplasm, resulting in MCM2 expression in the cytoplasm as well. On the other hand, p62 was normally expressed in the cytoplasm, while a large amount of p62 entered the nucleus in CHB patients. Therefore, combining our results, the interaction between p62 and MCM2 was scarce in normal conditions, while significantly enhanced in HBV infection. The interaction was attributed to the abilities of p62 and MCM2 to shuttle between the nucleus and cytosol during HBV replication, and the co-localization signal mainly concentrated in the nucleus (Fig. 6K). In HBV-infected mice, MCM2 expression was elevated, and ANXA4 depletion decreased it (Additional file 3: Fig. S10A). Meanwhile, ANXA4 expression also increased in hepatitis patients with different etiologies, especially in CHB patients (Additional file 3: Fig. S10B–C). More importantly, the MCM2 level was positively correlated with the expression of ANXA4 in CHB patients (Additional file 3: Fig. S10D). Therefore, these results suggest that ANXA4 silencing reduces the level of MCM2 by promoting its autophagic degradation, thereby enhancing HBV replication.

Fig. 6
figure 6

ANXA4 silencing enhances HBV replication by promoting autophagic degradation of MCM2. A Top DEGs in the DNA replication pathway. B, C Heatmap and volcano plot of the top DEGs in the DNA replication pathway. D, E ANXA4 and MCM2 levels in shCont- and shANXA4-transduced HepG2.2.15 cells. F ANXA4, p62, and MCM2 expression in HepG2.2.15 cells transduced with shANXA4 and treated with an autophagy inhibitor. G, H ANXA4 and MCM2 levels in shCont- and shANXA4-transduced HBV-infected HepG2-NTCP cells. I ANXA4, p62, and MCM2 expression in HBV-infected HepG2-NTCP cells transduced with shANXA4 and treated with an autophagy inhibitor. J Protein interactions were detected by immunoprecipitation in HepG2.2.15 cells and HBV-infected HepG2-NTCP cells. K p62 and MCM2 co-localization analysis in healthy individuals and CHB patients (bar = 50 µm). The values are presented as the means ± SEMs. * p < 0.05, ** p < 0.01, *** p < 0.001

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MCM2 inhibits HBV replication and viral protein expression in vivo and vitro

To demonstrate the direct role of MCM2 in HBV replication, we knocked down MCM2 (Fig. 7A–B, F–G) in HepG2.2.15 cells and HBV-infected HepG2-NTCP cells. Similar to the findings with ANXA4 knockdown, HBV replication and transcription were enhanced by MCM2 silencing (Fig. 7C–E, H–J). And ANXA4 downregulation-mediated HBV replication activity was attenuated by MCM2 overexpression (Fig. 7K–M, O–Q). In addition, MCM2 overexpression reversed HBsAg and HBeAg hypersecretion into the supernatant in ANXA4-deficient cells (Fig. 7N, R). Next, MCM2-overexpressed mice were further established with recombinant MCM2 (rMCM2) protein (Fig. 8A). The His tag labeled rMCM2 protein can be engulfed by the liver and hepatocytes (Fig. 8B). MCM2 overexpression decreased the levels of cccDNA, pgRNA, HBV DNA, HBV RNA, and the viral protein HBcAg and HBsAg in ANXA4-dificient mice (Fig. 8C–D, H–I). More importantly, MCM2 upregulation attenuated liver functional impairment (Fig. 8E). Analysis of HBV indexes in peripheral blood also suggested that MCM2 overexpression maintained the decrease in the HBV DNA, HBeAg, and HBsAg levels (Fig. 8F–G). Together, these results show that MCM2 could directly inhibit HBV replication and also reverse the increased HBV replication activity under ANXA4-deficient conditions. Overall, our study reveals that ANXA4 is a protective factor and that is elevated during HBV infection, reducing autophagic degradation of MCM2; in turn, the accumulated MCM2 inhibits HBV replication and alleviates liver injury. ANXA4 is expected to be a new target for the treatment and prognostic evaluation of individuals with CHB infection.

Fig. 7
figure 7

MCM2 inhibits HBV replication and viral protein expression in vitro. A–B, F–G MCM2 mRNA and protein levels were reduced in HepG2.2.15 cells and HBV-infected HepG2-NTCP cells treated with si-MCM2. C, H HBV DNA and HBV RNA levels in HepG2.2.15 cells and HBV-infected HepG2-NTCP cells were measured after MCM2 knockdown. D, I HBV DNA levels in the cell supernatant were measured. E, G The levels of HBsAg and HBeAg in the supernatant were measured by ELISA. K–L, O–P mRNA and protein levels of ANXA4 and MCM2 in HepG2.2.15 cells and HBV-infected HepG2-NTCP cells treated with shANXA4 and MCM2-Flag. M, Q HBV DNA and HBV RNA levels in HepG2.2.15 cells and HBV-infected HepG2-NTCP cells after shANXA4 and MCM2-Flag treatment. N, R The levels of HBsAg and HBeAg in the supernatant were evaluated. The values are presented as the means ± SEMs. * p < 0.05, ** p < 0.01, *** p < 0.001

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Fig. 8
figure 8

MCM2 overexpression attenuates HBV replication activity and liver injury in ANXA4-deficient mice. A Schematic overview of the experimental setup. B His tag labeled rMCM2 proteins can be engulfed by the liver and hepatocytes (bar = 50 µm). C Protein expression analysis of ANXA4, MCM2, HBsAg, and HBcAg in HBV-infected mice with AAV-shCont or AAV-shANXA4 treatment together with PBS or rMCM2 injection. D Relative ANXA4 mRNA expression, HBV RNA, HBV DNA, cccDNA, and pgRNA levels in above groups. E Measurement of serum AST, ALT, ALB, and TBA in the above mice. F Serum HBeAg and HBsAg were quantified by a chemiluminescent microparticle immunoassay in “shCont + HBV,” “shANXA4 + HBV,” and “shANXA4 + rMCM2 + HBV” group. G Serum HBV genomic DNA was also quantified in “shCont + HBV,” “shANXA4 + HBV,” and “shANXA4 + rMCM2 + HBV” mice. H, I IF of MCM2, HBcAg, and HBsAg expression (bar = 50 µm). The values are presented as the means ± SEMs. * p < 0.05, ** p < 0.01, *** p < 0.001

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Discussion

Annexins are multifunctional biological regulators involved in the inflammatory immune response, coagulation, and fibrinolytic processes, which is closely related to liver disease [22,23,24,25,26]. Based on the characteristic of cell membrane localization, annexin A family participates in viral invasion and host interaction. ANXA4 expression was elevated in COVID-19 patients and available for protective responses to this virus [11]. HBV is an enveloped virus with a partially double-stranded relaxed circular DNA genome of 3.2 kb [3,4,5]; the relationship between ANXA4 and HBV or HBV-related liver diseases is obscure. In this study, we found significantly elevated ANXA4 expression in patients with CHB compared to hepatitis patients with other etiologies, showing a link between ANXA4 and HBV infection. This phenomenon was verified in HBV-infected cell and mouse models, and we further revealed that ANXA4 can inhibit HBV replication and viral protein expression to alleviate liver injury. However, the increased HBsAg and HBeAg levels in the supernatant may be explained by the enhanced cell death [27], not only due to the activated HBV replication. Accordingly, cell death assay further confirmed that downregulation of ANXA4 in the cells infected with HBV did not increase cell death. Thus, the increase of HBsAg and HBeAg levels in the supernatant could be mainly explained by the enhanced HBV replication when ANXA4 was downregulated. The upregulated ANXA4 is important for the antiviral response and protection against the constant severity of HBV infection.

Autophagy is a catabolic process that clears aging and damaged proteins and organelles. Alteration of autophagy is involved in the protective reaction and innate and adaptive immune responses to viral infection [28,29,30,31]. As reported, the accumulation of autophagic vacuoles promoted hepatitis C virus replication and viral protein expression [32,33,34,35,36,37,38]. Actually, the relationship of HBV infection and autophagy induction has been reported. Accumulating evidence supports the close interaction between cellular autophagy and HBV infection [14, 39,40,41]. Firstly, HBV infection can directly activate autophagy pathways [39,40,41,42], and mechanistically, HBV infection-induced autophagosome formation and autophagy activation promoted viral DNA replication [43]. On the other hand, autophagy promoted HBV replication in vitro and vivo [44]. Transgenic mice carrying an overlength HBV DNA genome with liver-specific knockout of ATG5 confirmed an essential role of autophagy in HBV replication [15] and indicated the possibility of targeting the autophagic pathway for the treatment of CHB patients [40]. And inhibition of the autophagy-related gene Atg7 also inhibited HBV replication. These findings suggest a close link between autophagy and HBV replication. In our current study, we also explored the effects of autophagy on HBV infection and found that autophagy inhibition could directly suppress HBV replication. Annexins are multifunctional biological regulators involved in autophagy pathway and relate to liver diseases [26, 45,46,47]. Basing that, the invasion of HBV was accompanied by the increased expression of ANXA4, and ANXA4 could inhibit HBV replication and viral protein expression to alleviate liver injury. As we found previously, autophagy inhibition could directly suppress HBV replication, and we further revealed that 3-MA or CQ treatment both reversed the high expression of HBsAg and HBcAg resulting from ANXA4 downregulation, thereby inhibiting HBV replication. In conclusion, autophagy is identified as an important process in ANXA4-mediated alterations in HBV replication. Inhibition of ANXA4 enhances HBV replication and transcription by activating autophagy. Our findings demonstrate how ANXA4 intersects the autophagy pathway during HBV infection and emphasize that the ANXA4-mediated autophagy inhibition (concentrated in the inhibition of autophagic degradation) is important for HBV replication suppression to alleviate HBV infection process and liver injury.

Surprisingly, the DNA replication pathway was identified when ANXA4 was inhibited. As a DNA virus, HBV replication is associated with its transcription initiation and viral protein expression. MCM2, a participant of the DNA replication pathway, is important for DNA unwinding activity and genomic stability regulation, and abnormal MCM2 expression impairs DNA replication and cell cycle to induce disease processes [48,49,50,51,52]. Furthermore, HBV core protein can interact with MCM protein [53,54,55,56,57,58]. We found ANXA4 expression also increased in hepatitis patients with different etiologies, especially in CHB patients. More importantly, the MCM2 level was positively correlated with the expression of ANXA4. Further, we revealed that MCM2 expression was inhibited by ANXA4 downregulation and that MCM2 deficiency directly enhanced HBV replication. Similarly, MCM2 deficiency was found to increase the expression and secretion of cccDNA, HBV DNA, and HBeAg to enhance HBV replication activity [28, 29], while MCM2 overexpression attenuated HBV replication activity and alleviated liver injury induced by ANXA4 deficiency. The mechanism by which MCM2 inhibited HBV replication may derive from the regulation of nucleosome spacing to rearrange HBV small chromosomes [30].

We deeply explored the relationship among ANXA4, autophagy, and MCM2. The cargo protein p62 and autophagic marker LC3B are key regulators that play indispensable roles in the autophagic process. ANXA4 can competitively bind to p62 and LC3B to limit p62-LC3B interaction. Therefore, inhibition of ANXA4 enhances the p62-LC3B interaction to promote autophagy. We also found that MCM2 can directly bind to p62 but not ANXA4. MCM2 was transported by the substrate protein p62 for autophagy-dependent degradation, and ANXA4 downregulation increased the MCM2-p62 interaction and autophagic degradation of MCM2, activating HBV replication. In addition, we showed that autophagy induced by EBSS or rapamycin did not affect MCM2 degradation in hepatocytes without HBV infection, while activated autophagy promoted the autophagic degradation of MCM2 in HBV-infected cells. Moreover, ANXA4-regulated autophagy pathway did not affect the expression of MCM2 in the cells without HBV replication. The difference may be due to the fact that MCM2 can bind to autophagy protein p62 and be subsequently degraded in the presence of HBV infection, whereas this does not occur in the absence of HBV infection. Actually, MCM2 was located in the nucleus and rarely expressed in the normal liver. However, the expression of MCM2 in the nucleus increased significantly and partially transferred to the cytoplasm, resulting in MCM2 expression in the cytoplasm as well under HBV infection. It is reported that hypoxic replicon promoted chromatin bounding and nucleosolic MCM2, leading to a decrease in the cytosolic MCM2, while recovery of oxygen induced phosphorylation and diminution of chromatin bound MCM2, whereas cytosolic MCM2 increased [59], highlighting the important role of MCM2 located in the cytoplasm. On the other hand, p62 was normally expressed in the cytoplasm, while a large amount of p62 entered the nucleus in CHB patients. Phosphorylation is an important protein modification of p62; past studies showed that p62 shuttled continuously between nuclear and cytosolic compartments at a high rate [60], which was modulated by phosphorylation and aggregation of p62. Thus, p62 phosphorylation may lead to its transfer into the nucleus under CHB infection. Combining our results, the interaction between p62 and MCM2 was scarce in normal conditions, while significantly enhanced in HBV infection. The interaction was attributed to the abilities of p62 and MCM2 to shuttle between the nucleus and cytosol during HBV replication, and the co-localization signal mainly concentrated in the nucleus. In conclusion, ANXA4 inhibits autophagic degradation of MCM2 by obstructing the interaction of p62 and MCM2, and accumulated MCM2 thereby suppresses HBV replication. Meanwhile, in our results, we found that the transcription process of MCM2 was also partially regulated by ANXA4. Therefore, besides the analysis of protein degradation pathway, further exploration is needed on the full regulatory mechanism of ANXA4 on MCM2 expression and their synergistic effect on HBV replication.

From a clinical perspective for CHB and ANXA4, we firstly found that ANXA4 was also significantly increased in the serum of CHB patients and the ANXA4 level in serum was positively correlated with its expression in the liver. Importantly, hepatitis B viral protein expression was negatively correlated with ANXA4 levels in CHB patients, indicating that ANXA4 may inhibit HBV replication. In addition, CHB patients with high ANXA4 levels (> 8 ng/ml) showed increased sensitivity to interferon therapy, including higher cure rates, greater decline rate of HBsAg, and shorter clinical cure time. Through this work, we hope to predict the sensitivity of CHB patients to IFN treatment (including the possibility and time of cure, as well as the decrease in HBsAg) and to determine the prognosis and outcome of the untreated CHB patients by detecting their initial ANXA4 levels. Certainly, under the inhibition of HBV replication by IFN treatment, the expression level of ANXA4 may also decrease accordingly, and the specific role and detailed mechanism will be evaluated in our future work.

Conclusions

In conclusion, our work reveals the anti-HBV activity of ANXA4. ANXA4 expression is elevated under HBV attack, inhibiting autophagic degradation of MCM2 and thereby limiting HBV replication and transcription to alleviate liver injury and suppress the CHB infection process. ANXA4 also enhances the sensitivity of CHB patients to interferon therapy and is thus expected to be a new target for CHB treatment and prognostic assessment.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

3-MA:

3-Methyladenine

AAV:

Adeno-associated virus

AAV-HBV:

Recombinant adeno-associated virus containing HBV

AAV-shANXA4:

Recombinant adeno-associated virus containing shANXA4

AFP:

Alpha fetoprotein

ALB:

Albumin

ALD:

Alcoholic liver disease

ALT:

Alanine aminotransferase

ANXA4:

Annexin A4

AST:

Aspartate aminotransferase

BSA:

Bovine serum albumin

cccDNA:

Circular DNA

CHB:

Chronic hepatitis B

Co-IP:

Co-immunoprecipitation

CQ:

Chloroquine

DAPI:

4′,6-Diamidino-2-phenylindole

DCD:

Donation after cardiac death

DEGs:

Differentially expressed genes

DMEM:

Dulbecco’s modified Eagle’s medium

DMSO:

Dimethyl sulfoxide

DNBs:

DNA nanoballs

EBSS:

Earle’s balanced salt solution

ELISA:

Enzyme-linked immunosorbent assay

FBG:

Fasting blood glucose

FBS:

Fetal bovine serum

FT3:

Free triiodothyronine

FT4:

Free thyroxine

GC:

Genome copies

HBcAg:

Hepatitis B core antigen

HBeAg:

Hepatitis B e antigen

HBsAg:

Hepatitis B surface antigen

HBV:

Hepatitis B virus

HCV:

Hepatitis C virus

IF:

Immunofluorescence

IFN:

Interferon

IHC:

Immunohistochemical

IP:

Immunoprecipitation

KEGG:

Kyoto Encyclopedia of Genes and Genomes

MCM2:

Minichromosome maintenance complex component 2

MOI:

Multiplicity of infection

NAFLD:

Nonalcoholic fatty liver disease

NEUT#:

Absolute neutrophil count

ns:

Not significant

P/S:

Penicillin-streptomycin

p62:

Sequestosome 1

PBS:

Phosphate-buffered saline

PCA:

Principal component analysis

PEG:

Polyethylene glycol

pgRNA:

Pre-genomic RNA

RAPA:

Rapamycin

rMCM2:

Recombinant MCM2

RNA-seq:

RNA sequencing

RT-qPCR:

Quantitative RT-PCR

TB:

Total bilirubin

TEM:

Transmission electron microscopy

TSH:

Thyroid stimulating hormone

WB:

Western blotting

WBC:

Wide blood cell

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Acknowledgements

The authors thank all the participants of this study for their contribution. The authors also thank the reviewers for their helpful comments on this article.

Funding

This study was supported by grants from the Natural Science Foundation of China (No. 82070611), Natural Science Foundation of Guangdong Province (2020A1515010317), GuangDong Basic and Applied Basic Research Foundation (No. 21202104030000608 and 2021A1515220029), Guangzhou Science and Technology Plan Projects (202102010204 and 2023B03J1287), Sun Yat-Sen University Clinical Research 5010 Program (2020007 and 2018009), Transformation of Scientific and Technological Achievements Project of Sun Yat-sen University (No. 82000–18843236), Five-Year Plan of the Third Affiliated Hospital of Sun Yat-sen University (K00006 and P02421), Natural Science Foundation of Guangdong Province (2022A1515011056), National Natural Science Foundation of China (No. 82204447), and Natural Science Foundation for Youths Projects of Shandong Province (ZR2024QH275). This work was also supported by the Postdoctoral Fellowship Program of CPSF under Grant Number GZB20240394.

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Author notes

  1. Luo Yang, Xianzhi Liu, and Limin Zhen contributed equally to this work and share first authorship.

Authors and Affiliations

  1. Department of Infectious Diseases, The Third Affiliated Hospital of Sun Yat‐sen University, Guangzhou, Guangdong, China

    Luo Yang, Ying Liu, Lina Wu, Wenxiong Xu, Liang Peng & Chan Xie

  2. Guangdong Key Laboratory of Liver Disease Research, The Third Affiliated Hospital of Sun Yat‐sen University, Guangzhou, Guangdong, China

    Luo Yang, Xianzhi Liu, Ying Liu, Lina Wu, Wenxiong Xu, Liang Peng & Chan Xie

  3. Xiang’an Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China

    Xianzhi Liu

  4. Guangdong Cardiovascular Institute, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong, China

    Limin Zhen

  5. Department of Breast Surgery, The First Affiliated Hospital of Shandong First Medical University & Shandong Provincial Qianfoshan Hospital, Shandong Medicine and Health Key Laboratory of General Surgery, Jinan, Shandong, China

    Luo Yang

Authors
  1. Luo Yang
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  2. Xianzhi Liu
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Contributions

LY and XL designed and performed the experiments. LY and XL wrote the first draft of the manuscript and incorporated revisions. LZ collected liver samples for mRNA sequencing. YL, LW and WX participated in conducting the experiments. LP and CX guided the project and revised the manuscript. All authors contributed to the article and approved the submitted version.

Corresponding authors

Correspondence to Liang Peng or Chan Xie.

Ethics declarations

Ethics approval and consent to participate

The protocol for the study with clinical samples was approved by the Medical Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University (approval number: 20210228701). Informed consent was obtained from all participants involved in the study. The experimental animals were inspected and approved by the Institutional Animal Care and Use Committee of South China Agricultural University (approval number: 2022d051).

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All authors have given their consent for publication of this manuscript. We confirm that all necessary approvals have been obtained and that the manuscript does not violate any confidentiality agreements or rights.

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The authors declare no competing interests.

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Supplementary Information

Additional file 1. Primer sequences.

Additional file 2. Antibodies.

12916_2024_3724_MOESM3_ESM.docx

Additional file 3: Figures S1–S10. Fig. S1 Identification of ANXA4 by RNA-seq in liver tissues from patients with CHB and healthy liver tissues obtained from donors. Fig. S2 ANXA4 overexpression inhibits HBV replication and viral protein expression. Fig. S3 Establishment of the HBV-infected mouse model. Fig. S4 ANXA4 downregulation enhances HBsAg and HBcAg expression. Fig. S5 Serum ANXA4 level is associated with cure rate and time in CHB patients with IFN treatment. Fig. S6 ANXA4 upregulation suppresses autophagy and HBV replication. Fig. S7 Inhibition of autophagy could directly suppress HBV replication. Fig. S8 MCM2 is regulated by ANXA4. Fig. S9 The effects of autophagy activation on MCM2 expression. Fig. S10 The correlation of MCM2 and ANXA4 in HBV-infected mice and patients.

Additional file 4. Baseline characteristics of participants.

Additional file 5. Original blots.

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Yang, L., Liu, X., Zhen, L. et al. ANXA4 restricts HBV replication by inhibiting autophagic degradation of MCM2 in chronic hepatitis B. BMC Med 22, 521 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12916-024-03724-1

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BMC Medicine

ISSN: 1741-7015

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