Transcriptome sequencing chip data acquisition

With normal samples as controls, the chip datasets GSE36376 and GSE87630 were analyzed for differential gene expression using the “limma” package in R software, and the false discovery rate (FDR) method was used to correct the differential P values. Differentially expressed genes (DEGs) were obtained by screening with a significance level of FDR < 0.05 as the threshold for differential gene selection. The obtained DEGs were visualized using the “pheatmap” package in R for generating heatmap and volcano plot of expression data.

Intersectional gene screening

The genes associated with “HCC” were acquired from the GeneCards database. Subsequently, we utilized the “VennDiagram” package within the R software to identify the intersection between the differentially expressed genes found in the chip datasets GSE36376 and GSE87630 and the genes associated with HCC from the GeneCards database. These overlapping genes were then designated as candidate genes for subsequent analysis [42].

Prognostic analysis

Based on the FPKM format RNAseq data from LIHC project in TCGA database (https://portal.gdc.cancer.gov/), R software was used to perform differential expression analysis and prognosis-related gene selection on the intersection genes, with prognostic parameter setting type as Progress Free Interval (PFI). Subsequently, differential expression analysis of paired samples was performed on the top 5 genes with significant differences. Survival analysis, ROC curve visualization, independent prognostic analysis, and clinical correlation analysis were carried out using the “survminer” package (version 0.4.9) and the “survival” package (version 3.2–10) in R software, citing relevant literature. During the analysis, grouping was performed based on gene expression levels of 0–50% and 50–100%.

Enrichment analysis of functionalities

The chip datasets, namely GSE36376 and GSE87630, were subjected to identical preprocessing steps involving gene screening and data integration, a process executed using the “limma” package within the R software. Following the exclusion of normal samples, a differential expression analysis was conducted. For the subsequent functional annotation and pathway analysis, we harnessed the “ClusterProfiler” package within the R software. The Gene Ontology (GO) enrichment analysis encompassed three hierarchical levels: Biological Process (BP), Cellular Component (CC), and Molecular Function (MF), with a significance threshold set at P < 0.05. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was executed to identify cellular functions and signaling pathways primarily influenced by the differentially expressed genes, employing a notable enrichment cutoff of P < 0.05.

Cell culture and transfection

Human normal hepatic stellate cells LX-2 were purchased from ATCC (https://www.atcc.org/) and cultured in DMEM (Gibco, 11965092, USA) containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 environment. LX-2 cells were infected with HBV at 1012 U/L for 36 h [43].

Following HBV infection, LX-2 cells were categorized into distinct experimental groups based on various treatments: the sh-NC group (silencing lentivirus control group), sh-NAC1-1 group (silenced NAC1 slow virus group 1), sh-NAC1-2 group (silenced NAC1 slow virus group 2), oe-NC group (overexpression slow virus control group), oe-NAC1 group (overexpression slow virus NAC1 group), sh-NC + oe-NC group (silenced slow virus control + overexpression slow virus control group), sh-NAC1 + oe-NC group (silenced NAC1 slow virus + overexpression slow virus control group), and sh-NAC1 + oe-LDHA group (silenced NAC1 slow virus + overexpression slow virus LDHA group). The specific knockdown sequences are detailed in Table S1. Both the silencing and overexpression of NAC1 are mediated by lentivirus. The recombinant lentiviruses for gene silencing and overexpression were prepared and supplied by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). Cell transfection was carried out as follows: LX-2 cells in the logarithmic growth phase were harvested and resuspended at a concentration of 5 × 104 cells/mL. Subsequently, 2 mL of this cell suspension was seeded into each well of a 6-well plate, followed by an overnight incubation at 37 °C. In each well, recombinant lentiviruses at a concentration of 1 × 108 TU/mL, either silencing or overexpressing the target gene, were added. After 24 h of infection, the efficiency of GFP infection was assessed under a fluorescence microscope, and cells displaying robust effects were selected for subsequent experiments. Each experimental procedure was replicated three times for accuracy and consistency.

Co-cultivation system

HBV-infected or uninfected LX-2 cells were co-cultured with immune cells Jurkat. Jurkat cells were placed in the upper chamber of Transwell while LX-2 cells were placed in the lower chamber. This process was done to detect the impact of LX-2 cells on the apoptosis and proliferation of immune cells Jurkat [10].

ELISA detection

The assessment of HBV-DNA (CS11321, Shanghai ChunShi Biological Technology Co., Ltd.) and HBeAg (CR-018, Lantu Biotech., China) expression levels in the culture medium’s supernatant was performed employing an ELISA kit. In this procedure, both the sample and control samples were introduced into separate wells. Subsequently, an Ab-HRP complex was added and the mixture was incubated at 37°C for one hour, followed by five washes with PBST. Following the washing steps, 100 mL of substrate solution was added to each well and incubated for 15 min, after which the reaction was halted. The absorbance at 450 nm was quantified utilizing a microplate reader from BIO-RAD, USA [43].

Flow cytometry

To assess cell apoptosis, flow cytometry was employed as the detection method. Initially, the cells were washed three times with cold PBS. Subsequently, apoptosis detection was carried out using the Annexin V-FITC/PI apoptosis detection kit sourced from BD Pharmingen, USA. The cells were resuspended in 500 μL of binding buffer, and under conditions devoid of light, 5 μL of Annexin V-FITC and 5 μL of PI were added. The mixture was thoroughly mixed and then incubated for a duration of 15 min. Following this incubation period, cell apoptosis was quantified using a BD FACSCalibur flow cytometer [44].

Construction of a mouse model of liver cirrhosis

The C57BL/6-HBV transgenic mice were purchased from Beijing Weitongda Biotechnology Co., Ltd. (Beijing, China). The mice were raised under specific pathogen-free conditions at the animal facility of the Institute of Biophysics, Chinese Academy of Sciences, and all animal-related research was approved by the Institutional Animal Care and Use Committee (No. SZPH-AE-21018).

In this experimental study, mice were subjected to various treatments. The modeling group received Mouse CD137 monoclonal antibody (Clone 2 A, SRP0607, Sigma-Aldrich), while Mouse IgG (12-370, Sigma-Aldrich) served as the control. The CD137 antibody (Clone 2 A) was administered via intraperitoneal injection at a dose of 100 µg per week for seven consecutive weeks. Serum collection and monitoring of serum ALT levels were carried out regularly throughout the study. The mice were categorized into five distinct groups, each consisting of 12 mice. These groups were as follows: Control (mice treated with IgG), Model group (mice treated with CD137 monoclonal antibody Clone 2 A), sh-NC + oe-NC group (mice initially treated with control lentivirus and overexpression lentivirus for 3 days, followed by treatment with CD137 monoclonal antibody Clone 2 A), sh-NAC1 + oe-NC group (mice initially treated with NAC1 knockdown lentivirus and overexpression lentivirus control for 3 days, followed by treatment with CD137 monoclonal antibody Clone 2 A), and sh-NAC1 + oe-LDHA group (mice initially treated with NAC1 knockdown lentivirus and LDHA overexpression lentivirus for 3 days, followed by treatment with CD137 monoclonal antibody Clone 2 A). The specific knockdown sequence for sh-NAC1 was as follows: 5’-CCGGGCTGAACTTATCAACCAGATTCTCGAGAATCTGGTTGATAAGTTCAGCTTTTTTG-3’. The lentivirus used had a titer of 1 × 1011 PFU and was procured from Gima Biological. The lentivirus was administered via tail vein injection at an approximate volume of 50 μL per mouse, and this administration was carried out twice a week for a total duration of 7 weeks.

At day 21, liver tissue samples were harvested from six mice in each experimental group for comprehensive histological analysis. This analysis encompassed the assessment of pathological changes in liver tissue and collagen fibrosis. Furthermore, liver cell apoptosis within the tissue was examined using TUNEL staining. Additionally, the levels of hepatic fibrosis markers, namely hydroxyproline (ab222941, abcam, UK) and hyaluronic acid (ab2877991, abcam, UK), were quantified in the homogenized mouse liver tissue utilizing specific assay kits. Subsequently, at the 7-month time point, six mice from each group were selected for further evaluation. Liver morphology in these mice was closely observed, and liver nodules were scrutinized through H&E staining. The formula used for calculating liver tumor volume is as follows: tumor volume = ½ L*W^2, where L represents tumor length and W represents tumor width. Additionally, the expression of the CD8 protein within the liver tissue was assessed using IHC staining techniques [28].

Pathological histology analysis

Liver tissues were collected from each group of mice, and liver sections with a thickness of 5 μm were prepared and embedded in paraffin. Subsequently, these sections were subjected to observation of inflammatory reactions and analysis of liver collagen deposition/accumulation using H&E staining and Sirius Red staining. For H&E staining, the following protocol was followed: Hematoxylin and eosin staining was conducted using PT001 reagent (procured from Shanghai Bogu Biological Technology Co., Ltd., Shanghai, China) at room temperature for a duration of 10 min, followed by washing in running water for 30–60 s. Differentiation was achieved by immersing the sections in 1% hydrochloric acid alcohol for 30 s, followed by another washing step in running water and subsequent immersion in 1% eosin solution at room temperature for 1 min. Dehydration was performed using a gradient of alcohol (with concentrations of 70%, 80%, 90%, 95%, and 100%), with each concentration applied for 1 min. This was followed by clearing with xylene for 1 min. The sections underwent two rounds of transparency treatment with xylene I and xylene II, each lasting 1 min. Finally, the sections were mounted with neutral gum within a fume hood. The ensuing liver tissue morphological changes or structural characteristics were observed and documented using an optical microscope (BX50; Olympus Corp, Tokyo, Japan) [45].

Sirius Red staining: Liver tissue sections were stained with a saturated solution of 0.1% Sirius Red in 0.1% picric acid, and rapidly turned green after 1 h. The distribution of collagen protein was quantified by Image Pro Plus software (Media Cybernetics, Bethesda, MD) to measure the positive staining area of the tissue. Four consecutive images (×40) were taken for each section [38].

TUNNEL staining

To detect liver cell apoptosis, the TUNEL Apoptosis Detection Kit-DAB (Catalog No. abs50022, ABclonal Biotech Co., Ltd., Shanghai, China) was utilized. The specific procedure is as follows: Liver tissue sections were sliced at room temperature. These sections were then incubated with the TdT/nucleotide complex for a duration of 1 h. Following the incubation, the sections were washed with PBS. Nuclear labeling was performed using horseradish peroxidase and diaminobenzidine (DAB). Subsequently, counterstaining was carried out using Hematoxylin. Apoptotic cells were observed under an optical microscope, and images were captured. Positive apoptotic cells exhibited a brown staining in the nucleus. In each section, 5 randomly selected fields of view were assessed to count and detect apoptotic cells. The apoptosis rate was quantified as a percentage of positive cells, calculated by dividing the number of positive apoptotic cells by the total cell count and multiplying by 100% [46].

Immunohistochemistry

Following the fixation of liver tissues from each group of mice using formalin, tissue sections were obtained by embedding the tissues in paraffin. After deparaffinization, rehydration, and antigen retrieval through slicing, the tissues were subjected to incubation with primary antibodies against F4/80 (diluted at 1:5000, ab300421, Abcam, UK) and CD8 (diluted at 1:2000, ab217344, Abcam, UK) to assess the expression of the relevant proteins [47]. Subsequently, observations were made, and images were captured using an optical microscope (CX43, Olympus, Japan) to document the expression patterns and distribution of these proteins within the liver tissue sections.

Western blot

To extract total cellular protein from each experimental group, a protein extraction kit (BB-3101, Bestbio, Shanghai, China) was employed. The protein concentration in each sample was quantified using a BCA protein assay kit (Beyotime Biotechnology, P0012S, China) and adjusted with deionized water as necessary. Subsequently, a 10% SDS-PAGE gel (P0012A, Biomics Biotechnologies Co., Ltd., Shanghai, China) was prepared. Each well was loaded with 50 μg of protein sample, and electrophoresis was conducted at constant voltages of 80 V and 120 V for 2 h, as appropriate. The protein content was then transferred onto a PVDF membrane (ISEQ00010, Millipore, Billerica, MA, USA) using the wet transfer method, applying a constant current of 250 mA for 90 min. Following the transfer, the PVDF membrane was blocked by incubating it in TBST buffer containing 5% skim milk powder for 2 h. After discarding the blocking solution, the membrane was washed once with TBST before being exposed to primary antibodies overnight at 4°C (see Table S2 for antibody details). Subsequently, the membrane was washed three times with TBST for 10 min each. A goat anti-rabbit IgG antibody labeled with horseradish peroxidase (ab6721, Abcam, Cambridge, UK) was applied at a dilution of 1:2000 and incubated at room temperature for 1 h. Following this incubation, the membrane was washed three times with PBST for 10 min each. The membrane was then immersed in an ECL reaction solution (Western blotKLS0100, Millipore) for visualization, and the developed results were obtained by exposing the membrane in a dark box. For quantification, the relative gray value between the target protein band and the reference band, typically β-actin, was calculated, representing the relative expression level of the protein of interest.

RT-qPCR

Total RNA was extracted from the samples using TRIzol reagent (15596018, Invitrogen, USA). Subsequently, the High-Capacity cDNA Reverse Transcription Kit (4368813, Invitrogen, USA) was employed to reverse transcribe the extracted total RNA into cDNA, utilizing β-actin as the internal reference. For real-time PCR (RT-PCR) experiments, an ABI7500 real-time PCR instrument (Thermo, USA) was utilized in conjunction with the SYBR® Premix Ex TaqTM (Tli RNaseH Plus) kit (RR820A, TaKaRa, Japan). The PCR reaction mixtures underwent amplification using a real-time fluorescence quantitative PCR instrument from ABI, USA. To analyze the final data, the 2-ΔΔCt method was employed. The primer sequences utilized in these experiments were synthesized by Invitrogen and are detailed in Table S3.

Statistical analysis

All data analyses were conducted using SPSS 21.0 statistical software (IBM, USA). Continuous variables were expressed as mean ± standard deviation. The t-test was employed to compare data between two groups, whereas one-way analysis of variance (ANOVA) was utilized for comparisons involving multiple groups. A significance level of P < 0.05 was considered to indicate statistically significant differences.