Human patient samples
Human liver tissue and serum samples from healthy individuals and patients with liver cirrhosis were provided by the Zhongnan Hospital of Wuhan University. The tissue samples were immediately snap-frozen in liquid nitrogen upon collection to preserve their integrity. Blood samples were centrifuged to separate the serum, which was then aliquoted and stored at −80 °C for future analysis. All subjects provided written informed consent. The study, which included both liver tissue and serum samples, complied with the Declaration of Helsinki and Declaration of Istanbul and was approved by the Ethics Committee of Wuhan University (NO.WHU-LFMD-IRB2024036).
Establishment of the hepatic fibrosis model using S. japonicum or CCl4
Male BALB/c mice purchased from the Hubei Provincial Center for Disease Control and Prevention (Wuhan, China), 7–8 weeks old, weighing 22–25 g, were used. Oncomelania hupensis snails were provided by the Snail Ecological Station of Gongan County (Jingzhou, China). Liver fibrosis models were established in BALB/c mice through either percutaneous infection with 16 S. japonicum cercariae or intraperitoneal (i.p.) injection of CCl4 (Sigma-Aldrich; 10 ml/kg, diluted 1:4 in olive oil, filtered, twice weekly for 6 weeks). Recombinant human IL11 (rhIL11; UniProtKB: P20809, Genscript) was administered i.p. to mice at a dose of 100 mg/kg, once every two days. Mice were randomly assigned to groups (n = 6 per group) and included if healthy, with exclusion for severe distress or mortality during treatment. The investigators were blinded to the group allocation during the experiment. All animal experiments were approved by the Institutional Animal Care and Use Committee of Wuhan University (IACUC-AF048).
Construction and stereotactic injection of vectors for MCM7-knockdown or overexpression
Recombinant adeno-associated virus (AAV) vectors were obtained from Dr. Y.C. Xia (Wuhan University). An AAV-CMV vector (Addgene; Cat No.105530) was constructed to express the complete coding sequence of Mcm7 (AAV-MCM7 for Mcm7 overexpression), while empty vectors were used as negative controls. The recombinant plasmids were produced using a triple-transfection helper-free method and purified according to a previously published protocol [22]. Specifically, HEK293T cells (ATCC) were transfected with the AAV-CMV, AAV8 (Addgene; Cat No.112864), and AAV-helper (Addgene; Cat No.81070) plasmids in 150-mm dishes at 80% confluence. For Mcm7 knockdown, AAV-shCtrl (Addgene; Cat No.60958) vectors expressing a short hairpin RNA targeting Mcm7 mRNA (AAV-shMcm7) were constructed and verified by sequencing. AAV-shCtrl vectors served as negative controls. HEK293T cells were transfected with the AAV-shCtrl, AAV-DJ (Addgene; Cat No.130878), and AAV-helper (Addgene; Cat No.81070) plasmids. GFP was used as a reporter. Similarly, AAV-shShcbp1 and AAV-SHCBP1 vectors were constructed for Shcbp1 mRNA knockdown and overexpression, respectively, using the same methods described above for the Mcm7 experiments. Viral titers were determined by quantitative PCR. The final viral preparations were resuspended in sterile saline solution and had a titer of 1 × 1012 viral particles/ml.
Isolation of primary hepatocytes and HSCs
Primary hepatocytes and HSCs were isolated from the indicated mice through the portal vein after anesthesia, as previously described [23]. Briefly, the liver was perfused in situ with Ca2+-free Hank’s Balanced Salt solution for 15 min, followed by sequential perfusion with 100 mL of 0.2% pronase solution and 0.2% collagenase type IV (BioFroxx, Einhausen, Germany) until the liver appeared digested and pale in color. The resulting cell suspension was filtered through a 100 μm pore size nylon mesh, and then centrifuged at 50 × g. The pellet was collected for primary hepatocyte isolation, while the superannuate was further processed for primary HSC isolation using density gradient centrifugation. The isolated primary hepatocytes and HSCs were cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS).
Cell culture and treatment
The human hepatocyte cell line HepG2 and the human embryonic kidney cell line HEK293T were obtained from the American Type Culture Collection (ATCC). The human hepatic stellate cell line LX-2 was provided by Dr. Yong Wang (Nanjing Medical University), with its original source being ATCC. Additionally, the mouse hepatocyte cell line AML12 was utilized in this study. All the cell lines were authenticated by STR profiling and tested clean for mycoplasma contamination.
Cells were cultured in DMEM with 10% FBS. Recombinant human IL-1β (PeproTech, Rocky Hill, NJ, USA) and recombinant mouse IL-6 (PeproTech, Rocky Hill, NJ, USA) at a dose of 10 ng/ml were added to stimulate cells. Additionally, 10 μM XMU-MP-1 (MedChemExpress, Monmouth Junction, NJ, USA) was used to inhibit the Hippo signaling pathway, and 10 μM C188-9 (MedChemExpress, Monmouth Junction, NJ, USA) was used to inhibit the STAT3 signaling pathway. LX-2 cells and primary HSCs were treated with 10 μg/ml IL11 neutralizing antibody or IgG control (R&D Systems, Minneapolis, MN, USA) for 24 h.
Co-culture of HSCs with hepatocytes
Primary HSCs were isolated from normal mice and cultured overnight, while primary hepatocytes from liver fibrosis mice, with or without MCM7 overexpression, were isolated and cultured for 24 h. The conditioned medium from these primary hepatocyte cultures was collected, filtered through a 0.2-μm strainer, and then added to the primary HSC cultures. Additionally, HepG2 cells were transfected with an MCM7 overexpression vector or a control vector, then stimulated with IL-1β. The conditioned medium from these HepG2 cell cultures was co-cultured with the human HSC cell line LX-2 for 24 h. Lysates from the primary HSCs and LX-2 cells were then harvested for further experiments.
Quantitative reverse transcriptase PCR (qRT-PCR)
Total RNA was isolated from the cells using TRIzol reagent (Life, USA), following the manufacturer’s instructions. cDNA synthesis was performed using the HiScript II Q RT Super Mix for qPCR (Vazyme Biotech, Nanjing, China). The expression levels of target genes were measured by quantitative real-time PCR (qRT-PCR) using SYBR Green (Vazyme Biotech, Nanjing, China) on the Applied Biosystems 7300 Fast Real-time PCR System (USA). The relative RNA expression levels were calculated using the 2−ΔΔCt method, with GAPDH as the internal control. The primer sequences are listed in Supplemental Table 1.
RNA-Seq and data analysis
Total RNA was extracted from the samples using the RNeasy Kit (Qiagen, Hilden, Germany), and the RNA quality was assessed using an Agilent Bioanalyzer 2100. The mRNA was then purified using the KAPA mRNA Capture Kits (Roche, Basel, Switzerland), and cDNA libraries were prepared using the KAPA RNA HyperPrep Kits (Roche, Basel, Switzerland) at Kindstar Global (Wuhan, China). Equal amounts of cDNA library from each sample were pooled and sequenced on an Illumina HiSeq × platform, with 150-bp paired-end sequencing. The sequencing depth ranged from 85.567278 to 130.156120 million reads per sample, with a median of 113.913107 million reads. The sequencing reads were mapped to the GRCh38/GRCm39 genome assemblies using Hisat2 v2.1.0 with the default settings. The aligned reads were then converted to bigwig coverage files using reads per million. Genome annotations were extracted from the Ensembl GRCh38/GRCm39 Ens_96 database and used to count the reads with htseq-count v0.13.5. Significantly differentially expressed genes (DEGs) were identified using R (version 3.6.0) and the DESeq2 package, with criteria set at an adjusted p-value (padj) <0.05 and an absolute log2 fold change (log2FC) ≥1. Functional enrichment analysis of the identified DEGs was performed using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases.
Immunoprecipitation mass spectrometry assay
Stable HL7702 hepatocyte cell lines expressing MCM7 (pHAGE-MCM7) were cultured for 48 h. The cells were then dissolved in a cell suspension containing: 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 340 mM sucrose (~12% w/v), 10% (v/v) glycerol, 0.5 mM DTT, protease inhibitor cocktail (1:100), and 10 mM sodium butyrate (HDAC inhibitor from a 1 M stock solution). The samples were treated with an equal volume of 0.2% Triton, resulting in a final Triton concentration of 0.1%, and a final CaCl2 concentration of 1 mM. Anti-MCM7 antibodies were added to the samples, which were then incubated overnight at 4 °C. Subsequently, A/G-agarose beads were added and incubated for 4 h. The beads were washed twice with PBST, and 100 μl of elution buffer mixed with SDS loading buffer was added to each sample. Subsequently, 15 μl of supernatant was collected for gel running, and silver staining was used for detection, whereas the remaining samples were stained with Coomassie blue. The whole gel was cut into pieces, washed three times with H2O, and destained using a solution containing 35% ACN and 50 mM NH4HCO3. After dehydration, reduction and alkylation of cysteines, the gel was digested in 50 mM NH4HCO3 solution with modified trypsin at 37 °C overnight. The resulting tryptic peptides were analyzed on a Q Exactive HF-X mass spectrometer coupled with an Easy-nLC 1200 system (Thermo Scientific, USA) with a 60 min gradient. The mass spectrometer was operated in data-dependent acquisition mode with full scans (R = 60 K, AGC = 3e6, max IT = 20 ms, scan range = 300–1800 m/z) followed by 25 MS/MS scans (R = 15 K, AGC = 2e5, max IT = 50 ms). All MS/MS spectra were analyzed by pFind software (version 3.0.11) 2 against the human protein database combined with the reverse decoy database and common contaminants. Trypsin digestion allowed up to two missed cleavages, and an open-search algorithm implemented in pFind was employed for data analysis. The precursor and fragment ion mass tolerances were 20 ppm and 20 ppm, respectively. Minimum peptide length was set at 6, while the estimated false discovery rate threshold for peptide and protein was specified at maximum 1%. The mass spectrometry analysis for SHCBP1 was performed using the same protocol. The antibodies used in this study are listed in Supplemental Table 3.
Western blot
Total proteins were extracted from the tissues or cells using pre-chilled RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors (Thermo Scientific, USA). The protein concentration was determined using a Bicinchoninic Acid Protein Assay Kit (Thermo Scientific, USA). Equal amounts of the protein samples were separated by 10% SDS-PAGE and then transferred to 0.45 μm PVDF membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk in TBST for 1 h, and then incubated with the corresponding primary antibodies overnight at 4 °C. After three washes with TBST, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Following three additional TBST washes, the membranes were incubated with ECL Western blot Substrate (Bio-Rad, Hercules, CA, USA) and exposed to X-Ray Super RX Films (Fujifilm, Tokyo, Japan) for detection. The antibodies used in this study are listed in Supplemental Table 3.
Co-Immunoprecipitation (Co-IP)
Cells were lysed in cold IP buffer (150 mM NaCl, 25 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.5% Nonidet P-40) containing a protease inhibitor cocktail. The cell lysates were then centrifuged at 12,000 × g for 30 min. The obtained cell lysates were first pre-cleared with protein A/G-agarose beads (Smart-Lifesciences, Changzhou, China) at 4 °C for 6 h. Subsequently, the pre-cleared lysates were incubated with an anti-MCM7 antibody or an isotype-matched control IgG overnight at 4 °C. MCM7 and its associated proteins were then precipitated by incubating the lysates with pre-equilibrated protein A/G-agarose beads at 4 °C for 6 h. The immunoprecipitates were thoroughly washed with cold PBST, heated in 1× loading buffer at 95 °C for 5 min, and then analyzed by immunoblotting. The precipitates were then washed and analyzed by immunoblotting. The antibodies used in this study are listed in Supplemental Table 3.
GST/His pull-down assay
Expression of recombinant GST-tagged truncated MCM7 fragments and His-tagged serial truncations of SHCBP1 proteins were induced in BL21 cells at 28 °C for 24 h by 0.1 mM isopropyl β-D-1-thiogalactopyranoside. Bacterial cells were lysed using a lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.5% Triton X-100. Recombinant proteins were then purified using Glutathione Sepharose 4B beads (Smart-Lifesciences, Changzhou, China) and HisPur™ Ni-NTA Resin (ThermoFisher, Waltham, MA, USA). Protein concentrations were determined by measuring their optical density absorbance at 280 nm. The 100 µl GST-tagged truncated MCM7 fragments were mixed with 100 µl His-tagged full-length SHCBP1 as well as His-tagged serial truncations of SHCBP1 with GST-tagged MCM7 in 0.5 ml binding buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.5% Triton X-100). The binding reaction was performed with Glutathione Sepharose 4B beads and HisPur™ Ni-NTA Resin overnight at 4 °C, and the beads were subsequently washed three times with the binding buffer. Proteins were analyzed by Western blot using GST and His antibodies. The antibodies used in this study are listed in Supplemental Table 3.
Chromatin Immunoprecipitation (ChIP)
ChIP was performed as previously described [24]. Briefly, the cells were cross-linked with 1% paraformaldehyde for 10 min, and the reaction was quenched with glycine for 5 min. After cell lysis, the fixed chromatin was fragmented to 100–500 base pair DNA fragments using Diagenode Bioruptor Plus non-contact sonication device (Diagenode, Liège, Belgium). The DNA fragments were then immunoprecipitated using a STAT3 antibody and Protein A/G beads. The precipitated DNA samples were subsequently analyzed by quantitative PCR (qPCR). The antibodies used in this study are listed in Supplemental Table 3.
Luciferase reporter assay
HepG2 cells were seeded in a 24-well plate at a density of 1 × 105 cells per well, 24 h prior to transfection. The pGL3-basic reporter vectors containing the IL11 promoter fragment were then transfected using 1 μL of Neofect™ DNA Transfection Reagent (Neofect, Beijing, China), following the manufacturer’s instructions. After 24 h of transfection, the cells were harvested and lysed using reporter lysis buffer (Promega, Madison, WI, USA). Luciferase activities were measured using a Luciferase Assay System (Promega, Madison, WI, USA), and the firefly luciferase activities were normalized to the Renilla luciferase activities.
Histology and immunohistochemistry (IHC)
The liver samples were fixed with 4% paraformaldehyde and embedded in paraffin. The sectioned tissues were stained with Mayer’s hematoxylin and eosin (H&E) to measure the size of hepatic granulomas using a calibrated measuring eyepiece. Additionally, the tissues were stained with Masson’s trichrome to determine the extent of fibrosis. For the immunohistochemistry analysis, the liver sections (4 μm) were deparaffinized in xylene and hydrated using a graded ethanol-deionized water series. Antigen retrieval was performed using an Ethylene Diamine Tetraacetic Acid (EDTA) repair solution via microwave treatment. The sections were subsequently incubated with 3% hydrogen peroxide (H2O2) for 10 min at room temperature, followed by incubation with primary antibodies against MCM7, SHCBP1, COL1A1, IL11, and α-SMA. Next, the sections were incubated with a horseradish peroxidase-labeled secondary antibody at room temperature for 1 h. The immunoreactive signals were developed using a DAB reagent solution, and the nuclei were counterstained with hematoxylin solution. The stained sections were scanned and visualized using an Aperio Versa 8 system (Leica Biosystems, Wetzlar, Germany). For each group, whole-slide imaging was conducted on distinct tissue sections, with random fields of view selected for analysis. The positive areas were calculated and quantified using ImageJ software. The antibodies used in this study are listed in Supplemental Table 3.
Immunofluorescence histochemical analysis
Liver tissues were fixed overnight in 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 4 μm. The liver sections were then blocked in a solution containing 10% FBS and 0.1% Triton-100 at 4 °C for 2 h. Subsequently, the sections were incubated with primary antibodies against MCM7, Albumin, α-SMA, and F4/80 at 4 °C for 24 h. After washing, the sections were incubated with fluorescent-labeled secondary antibodies at 4 °C for 2 h. The stained sections were then scanned using a TCS SP8 STED CW confocal microscope (Leica Microsystems, Wetzlar, Germany). The immunofluorescence signals were quantified using ImageJ software. The antibodies used in this study are listed in Supplemental Table 3.
Enzyme-linked immunosorbent assay (ELISA)
The levels of IL11 in human and mouse serum samples were determined using the human IL11 ELISA Kit (CHE0010-096, 4A Biotech, Beijing, China) and the mouse IL11 ELISA Kit (CME0007-096, 4A Biotech, Beijing, China), respectively. The analyses were performed in accordance with the manufacturers’ instructions provided with the ELISA kits.
Measurement of hepatic hydroxyproline content
The hepatic hydroxyproline content was quantified using a hydroxyproline assay kit (A030-2, Jiancheng Bioengineering Institute, Nanjing, China), following the manufacturer’s instructions. Data were provided as μg·100 mg−1 wet liver tissue.
Statistics
All data are presented as the mean ± standard deviation (SD) from at least three independent experiments. The investigators were blinded to the group allocation during when assessing the outcome. The sample size was chosen to ensure adequate power to detect a prespecified effect size. Statistical analysis was performed using a 2-tailed Student’s t-test or 1-way or 2-way ANOVA, as appropriate. The correlation coefficient (r) was determined using Spearman’s rank correlation analysis. A p-value less than 0.05 was considered statistically significant.
