BNC1 inhibits the development and progression of gastric cancer by regulating the CCL20/JAK-STAT axis

Clinical samples

Authorization for the procurement and use of patient specimens for this study was received from the Ethics Committee of The Affiliated Hospital of North Sichuan Medical College (Approval Number: 2024ER463-1). This investigation was exempted from obtaining informed consent as it solely entailed immunohistochemical (IHC) examination of archived pathological samples and used anonymized data extracted from the hospital’s electronic medical records and databases, ensuring minimal risk to participants. A total of 61 matched pairs of gastric carcinoma tissues and corresponding adjacent normal tissues were acquired for IHC analysis. The pathological characteristics of these 61 gastric cancer cases are detailed in Table S1.

Cell lines and cell culture

The GES-1 normal human gastric epithelial cell line was obtained from GuangZhou Jennio Biotech Company. The GC cell lines HGC-27, SNU-1, and AGS were sourced from Shanghai Min Jin Biotechnology Company. The MKN-28 and MGC-803 GC cell lines were acquired from cells maintained at the Institute of Tissue Engineering and Stem Cells, Nanchong Central Hospital. For cell culture, GES-1, MKN-28, MGC-803, and SNU-1 cells were maintained in RPMI-1640 medium (Gibco). HGC-27 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco), and AGS cells were cultured in F-12K medium (Gibco). All media were supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂. To ensure the authenticity of the cell lines and prevent cross-contamination, short tandem repeat (STR) profiling was performed on all cell lines prior to experimentation.

siRNAs and transfection

The small interfering RNAs (siRNAs) targeting CCL20 (si-CCL20#1 through si-CCL20#4) and the negative control siRNA (si-NC) were procured from GenePharma. Transfections were performed using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer’s protocol. Detailed sequences of the siRNAs are provided in Table S2. The experimental procedures were conducted in strict accordance with the product manuals provided by GenePharma (https://www.genepharma.com/en/).

Quantitative RT-PCR

Forty-eight hours post-transfection, or upon establishment of stable GC cell lines, total RNA was extracted using the FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme Biotech Co., Ltd). Genomic DNA was removed and complementary DNA (cDNA) synthesis was performed with the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA Wiper) from the same supplier. Quantitative PCR amplification of the cDNA was conducted using the ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd; https://www.vazyme.com/). Primer sequences used for qPCR were synthesized by Sangon Biotech (Shanghai, China) and are detailed in Table S3. Relative gene expression levels were determined using the 2^−ΔΔCt method, with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) serving as the internal control.

ELISA validation

The stable BNC1-overexpressing MKN-28 and AGS cell lines were used to perform the ELISA analysis using the Human MIP-3α/CCL20 (C-C motif chemokine ligand 20) QuickTest ELISA Kit (QT-EH0231; Wuhan Fine Biotech Co., Ltd). Assays and analyses were conducted according to the manufacturer’s protocol.

Stable cell line construction

Lentiviral vectors engineered to overexpress BNC1 and confer puromycin resistance were procured from Shanghai GenePharma Co., Ltd. Prior to establishing stable cell lines, the minimum lethal concentration (MLC) of puromycin was determined by treating GC cells (MKN-28, MGC-803, and AGS) with increasing concentrations of puromycin (ranging from 0.5 to 5.0 µg/mL) over a three-day period. The MLC was identified as the lowest concentration of puromycin that resulted in complete cell death within this timeframe, which was found to be 1.0 µg/mL. Following transduction, the culture medium was refreshed with 10% FBS 24 h post-infection. After 72 h, selection commenced by introducing 10% FBS medium supplemented with 1.0 µg/mL puromycin. Stable gastric cancer cell lines overexpressing BNC1 were established within one week. The specific sequences used in the lentiviral constructs are detailed in Table S2.

Immunohistochemistry

IHC analysis was performed on 61 pairs of formalin-fixed, paraffin-embedded GC tissues from clinical sources and four pairs of paraffin-embedded subcutaneous GC tissue sections from animal experiments. The primary antibodies used were: BNC1 polyclonal antibody (DF8812, 1:150; Jiangsu Family Biology Research Center, Ltd), CCL20 polyclonal antibody (DF2238, 1:100; Jiangsu Family Biology Research Center, Ltd), Ki-67 (ready-to-use; Fuzhou Maxin Biotechnology Development Co., Ltd), and Her-2 polyclonal antibody (FNab03833; Wuhan Fine Biotech Co., Ltd). Two pathologists independently evaluated the IHC staining using a scoring system that combined staining intensity and the percentage of positive cells, with the final score being the product of these two parameters, ranging from zero to nine points. The pathologists were blinded to both the clinical data of the specimens and the specific antibodies used during their assessments. The detailed scoring criteria were as follows: the proportion of positive cells was rated as 0 (0–25%), 1 (25–50%), 2 (50–75%), or 3 (greater than 75%); staining intensity was categorized as 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellow), and 3 (strong staining, brown). The scoring results are presented in Tables S1 and S5.

Colony formation assays

GC cells were harvested during the logarithmic growth phase and prepared as a single-cell suspension. Cells were seeded into six-well culture plates at a density of 1,000 cells per well, with each well containing two mL of culture medium supplemented with 10% FBS. The cultures were maintained at 37 °C in a humidified atmosphere with 5% CO₂, and the medium was refreshed every three days. After an incubation period of 14 days, allowing for colony development, the cells were gently washed twice with phosphate-buffered saline (PBS) to remove residual medium. Next, the colonies were fixed by adding one mL of 20% methanol to each well and incubating at room temperature for 30 min. Following fixation, the methanol was aspirated, and one mL of 0.2% crystal violet staining solution was added to each well with a staining duration of three minutes at room temperature. Excess stain was removed by rinsing the wells gently with distilled water, and the plates were air-dried at room temperature. For colony quantification, images of the stained colonies were captured using a high-resolution digital camera. The acquired images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Dual luciferase reporter assays

To study the influence of BNC1 on CCL20 promoter activity, the researchers engineered the overexpression construct pCDNA3.1-BNC1 and the reporter plasmid pGL3-CCL20pro. AGS cells were seeded into 24-well plates at a density of 5 × 10⁴ cells per well and cultured overnight in F12K medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO₂. The following day, cells were co-transfected with 800 ng of pGL3-CCL20pro and 200 ng of either pCDNA3.1 (empty vector) or pCDNA3.1-BNC1 using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol. After 48 h of transfection, cells were lysed with 100 µL of Passive Lysis Buffer (Promega) per well. Next, 20 µL of the lysate was mixed with 100 µL of Luciferase Assay Reagent II to measure Firefly luciferase activity, followed by the addition of 100 µL of Stop & Glo Reagent to assess Renilla luciferase activity using the Dual-Luciferase Reporter Assay System (Promega) as per the manufacturer’s instructions. Luminescence was quantified with a GloMax 20/20 luminometer (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity to control for transfection efficiency.

Western blot analysis

Proteins were extracted using RIPA lysis buffer supplemented with protease inhibitors, PMSF, and phosphatase inhibitors. The extracted proteins were separated on a 10% SDS-PAGE gel prepared using the Omni-Easy™ One-Step PAGE Gel Preparation Kit (Epizyme Biotech). Following electrophoresis, proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% nonfat dry milk for one hour at room temperature and then incubated overnight at 4 °C with the following primary antibodies: BNC1 (PA5-66883, 1:2000; Invitrogen), JAK2 (R013499, 1:500; Shanghai Epizyme Biomedicine Technology Co., Ltd), STAT3 (10253-2-AP, 1:2000; Proteintech), p-STAT3 (28945-1-AP, 1:1000; Proteintech), BCL-2 (12789-1-AP, 1:2000; Proteintech), and BAX (50599-2-Ig, 1:2000; Proteintech). After four washes with phosphate-buffered solution with Tween 20 (PBS-T), the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:1000) or HRP-conjugated goat anti-mouse IgG (1:1000). Signal detection was performed using an enhanced chemiluminescence (ECL) reagent (Bio-Rad, Hercules, CA, USA), and images were captured using a gel imaging system or developed on X-ray film in a darkroom. Quantitative analysis of band intensity was conducted using ImageJ software (National Institutes of Health).

Flow cytometry

To evaluate apoptosis, 2 × 10⁵ MKN-28 and AGS cells overexpressing BNC1 were harvested. The cells were centrifuged at 300× g for five minutes, the supernatant was removed, and the pellet was washed twice with PBS. After the final wash, cells were resuspended in 500 µL of 1 × Annexin V Binding Buffer. Next, 5 µL of Annexin V-FITC and five µL of propidium iodide (PI, 50 µg/mL) were added. The mixture was gently vortexed and incubated in the dark at room temperature for 20 min. Flow cytometry analysis was then performed immediately.

For cell cycle analysis, approximately 4 × 10⁵ MKN-28 and AGS cells stably overexpressing BNC1 were collected. The cells were washed twice with cold PBS and resuspended in 0.3 mL of PBS. To this suspension, 1.2 mL of pre-chilled absolute ethanol (−20 °C) was added while vortexing, and the cells were fixed at −20 °C for at least one hour or overnight. Post-fixation, cells were centrifuged at 300× g for five minutes, the supernatant was discarded, and the pellet was resuspended in one mL of PBS and incubated at room temperature for 15 min. After another centrifugation and supernatant removal, 100 µL of RNase A reagent was added to fully resuspend the cells, followed by a 30-minute incubation in a 37 °C water bath. Next, 400 µL of PI reagent (50 µg/mL) was added, mixed thoroughly, and the cells were incubated in the dark at 2–8 °C for 30 min. Stained cells were immediately analyzed using a FACSCalibur flow cytometer (BD Biosciences), and data were processed with FlowJo software (BD Biosciences). All reagents used in these procedures were obtained from Elabscience Biotechnology Co., Ltd.

Xenograft model

Athymic nude mice were purchased from Chengdu GemPharmatech Biotechnology Co., Ltd. The mice were housed and cared for by trained professionals at the Animal Experimentation Center of North Sichuan Medical College. The animals were kept in a controlled environment with a temperature of 22 ± 2 °C and a 12-hour light/dark cycle. Each cage housed three to five mice with ad libitum access to food and water. A total of 20 animals were used for the subcutaneous tumor model. A random sequence generation method was used to allocate the animals into control and experimental groups with 10 mice in each group. All experimental mice were six-week-old male nude mice weighing approximately 20 g. After one week of acclimatization, the athymic nude mice were injected subcutaneously with 2 × 10⁶ AGS/NC or AGS/OE-BNC1 cells. Mouse weight and tumor volume were monitored every three days. Measurements were conducted by two individuals using the same caliper and weighing scale, with each researcher blinded to the other’s measurements. The final values were recorded as the average of the two independent measurements. Tumor size was calculated using the following formula: tumor volume (mm³) = width × length × (width + length) × 0.5. Animals were euthanized via intraperitoneal injection of euthanasia agents following predefined humane endpoints or experimental termination criteria. Humane endpoints included: (1) a 25% reduction in body weight, (2) complete loss of appetite for 24 h, or (3) inability to stand for 24 h due to weakness or an extreme reluctance to stand. Animals were also euthanized at experimental termination points, which included the end of the observation period (21 days post-injection) or a maximum of eight weeks post-treatment. Tumors were isolated upon euthanasia for photographing, IHC analysis, and hematoxylin and eosin (H&E) staining. If the subcutaneous tumor model failed to establish successfully or if animal mortality occurred during the experiment, both resulting in measurement failure, the corresponding data was excluded from the analysis. Animal studies were carried out according to the protocols approved by the ethics committee of the Institutional Animal Care and Use Committee of the North Sichuan Medical College (IACUC approval number: NSMC2024102).

Migration and invasion assays

The cells from each group were harvested through trypsinization, subjected to centrifugation, and rinsed twice with serum-free medium to eliminate any remaining serum. For the migration assay, cells were resuspended in serum-free medium at a concentration of 2 × 10⁵ cells/mL. A 100 µL aliquot of this suspension was added to the upper chamber of each Transwell insert, while the lower chamber was filled with medium supplemented with 10% serum. After 48 h of incubation, the upper chambers were removed, and cells on the membrane’s upper surface were gently wiped away using a moistened cotton swab. The membranes were then fixed in 20% methanol and stained with 0.1% crystal violet for ten minutes. Cells that had migrated to the lower surface were visualized, photographed, and counted under a light microscope at 100 × magnification. For the invasion assay, the procedure mirrored that of the migration assay with the following modifications: the upper chambers were pre-coated with Matrigel, and the cells were resuspended at a density of 4 × 10⁵ cells/mL. All subsequent steps were conducted as described for the migration assay. Quantification of migrated and invaded cells was performed using ImageJ software.

Wound healing test

Cells stably expressing the lentiviral construct were seeded into six-well plates. After ensuring complete adherence, a sterile pipette tip was utilized to create uniform linear scratches in each well. Wound closure was monitored by capturing images at zero and 72 h using an inverted microscope. The migration rate was determined using the following formula: Scratch healing rate (%) = [(initial scratch width − scratch width at 72 h)/initial scratch width] × 100.

Cell counting kit-8 method

Cells stably expressing the lentiviral construct were harvested, enzymatically dissociated, and resuspended in culture medium. Following cell counting, 3,000 cells per well were plated into 96-well plates. Cell proliferation was evaluated at 24, 48, 72, and 96 h using the Cell Counting Kit-8 (CCK-8) assay (Vazyme Biotech Co., Ltd) by adding 10 µL of CCK-8 reagent to each well and incubating for one hour. Absorbance at 450 nm was measured with a microplate reader to determine cell viability.

Statistical analysis

Statistical analyses were conducted using GraphPad Prism version 9.0. Data were expressed as the mean ± standard deviation (SD) from a minimum of three independent experiments. Group comparisons were performed using one-way analysis of variance (ANOVA) while differences between two groups were assessed with Student’s t-test. Statistical significance was defined as P < 0.05, with significance levels denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Clinical relevance analysis for basonuclin 1

To investigate the role of the novel transcription factor basonuclin 1 (BNC1) in gastric cancer, IHC staining was performed on GC tissues and adjacent normal tissues randomly collected from 61 patients. BNC1 expression was predominantly nuclear and was significantly lower in cancer tissues compared to adjacent normal tissues (Figs. 1A, 1B). Additional analyses using patient clinical information (Table S1) revealed no significant correlation between BNC1 expression and tumor differentiation or patient gender. However, a significant relationship was observed between BNC1 expression and both lymph node metastasis and pathological stage (Figs. 1C–1E and Figs. S1A, S1B). BNC1 expression decreased progressively with increasing numbers of metastatic lymph nodes and advancing pathological stages. These findings suggest that reduced BNC1 expression is associated with tumor progression and metastasis in gastric cancer. Consequently, BNC1 may serve as a potential molecular marker for clinical diagnosis and is closely linked to the development and progression of gastric cancer.

Overexpression of BNC1 suppresses development of gastric cancer cells

To investigate the functional role of BNC1 in gastric cancer cells, its expression was assessed in the normal gastric epithelial cell line GES-1 and five gastric cancer cell lines—HGC-27, SNU-1, MGC-803, MKN-28, and AGS—using quantitative real-time PCR (qRT-PCR) and Western blot analyses (Figs. 2A–2C). Notably, the SNU-1 cell line is not suitable for conducting wound healing assays. As such, MGC-803 and AGS cell lines, which showed the most significant differences in BNC1 expression, were initially chosen for functional assays. However, modulation of BNC1 in the MGC-803 cell line did not result in significant biological changes (Figs. S2A, S2B). Therefore, MKN-28 and AGS cell lines were used for further experiments. Using a lentiviral system, stable BNC1-overexpressing MKN-28 and AGS cell lines were successfully established and confirmed by qRT-PCR and Western blot analyses (Figs. S2C–S2E). Compared to control cells, BNC1 overexpression significantly inhibited cell proliferation (Figs. 2D–2F). Transwell assays and wound healing assays demonstrated that the migratory and invasive capabilities of BNC1-overexpressing cells were markedly reduced (Figs. 2G–2J). Flow cytometry revealed that BNC1 overexpression induced G1 phase cell cycle arrest (Fig. 2K, Fig. 2L). Additionally, apoptosis assays indicated that BNC1 overexpression resulted in a significant increase in apoptotic cells (Figs. 2M–2P). These findings suggest that BNC1 acts as a tumor suppressor in gastric cancer cells.

BNC1 suppresses proliferation and metastasis of gastric cancer cells in vivo

To further investigate the function of BNC1 in nude mice, an AGS cell stable strain that overexpressed BNC1 using lentivirus was constructed. Subcutaneous tumor model experiments were carried out, and the results showed that, after overexpressing BNC1, tumor volume and tumor weight were significantly reduced (Figs. 3A–3D) and the nuclear-cytoplasmic ratio of tumors was also significantly reduced (Fig. 3E). Further IHC analysis was conducted to assess the proliferation and expression of metastasis markers. The results showed that the expression levels of Ki-67 and Her-2 were markedly lower in the BNC1 overexpression group compared to the control group (Figs. 3F–3H). These findings corroborate the in vitro results and imply that BNC1 overexpression suppresses tumor growth and metastasis in vivo.

BNC1 negatively regulates CCL20

Transcription factors specifically bind to DNA sequences to regulate gene expression. To identify downstream target genes of BNC1, AGS stable cell lines overexpressing BNC1 were established. Total RNA was extracted, purified, and used to construct libraries for sequencing on the Illumina platform. Transcriptome sequencing revealed a predominant downregulation of gene expression upon BNC1 overexpression, suggesting a significant negative regulatory effect. Clustering analysis further underscored this trend (Fig. 4A). Applying criteria of log₂FC <−1.5 and P < 0.05 to the transcriptome data identified 56 downregulated genes. To refine the search for key target genes, the GEPIA2 online bioinformatics tool (Tang et al., 2019) was used to download 3,742 highly expressed genes in gastric cancer from The Cancer Genome Atlas (TCGA) database. The intersection of these two gene sets yielded five key genes, as illustrated in the volcano plot (Figs. 4B, 4C). The qPCR validation showed that PTPRC, SERPINB9, and RAC2 had low expression in gastric cancer cells (Figs. 4D–4F), whereas only CCL20 and BANK1 were highly expressed (Figs. 4G, 4H). Consequently, further investigation focused on CCL20 and BANK1. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed significant enrichment in the chemokine signaling pathway (Fig. 4I), suggesting that CCL20 is a downstream target of BNC1. Subsequent qPCR and ELISA assays confirmed that overexpression of BNC1 led to downregulation of CCL20 at both mRNA and protein levels (Figs. 4J, 4K). Due to the absence of BNC1 in the JASPAR database (https://jaspar.elixir.no/tfbs_extraction/), the closely related BNC2 was used to predict potential binding sites on the CCL20 promoter (Table S4). The AlphaFold3 predictions suggested a high likelihood of interaction between BNC1 and these sites, implying that BNC1 acts as a transcriptional repressor of CCL20 (Fig. 4L). This hypothesis was validated by dual-luciferase reporter assays, which demonstrated that BNC1 negatively regulates CCL20 promoter activity (Fig. 4M). These findings indicate that BNC1 negatively regulates CCL20 expression in gastric cancer cells.

CCL20 promotes development of gastric cancer cells

This study established that BNC1 functions as a tumor suppressor in gastric cancer cells and negatively regulates its target gene, CCL20. A series of experiments was conducted to elucidate the biological role of CCL20 in GC progression. SiRNA technology was used to effectively silence CCL20 expression in gastric cancer cell lines (Figs. S3A, S3B). The downregulation of CCL20 resulted in a significant reduction in cell proliferation as determined by CCK8 and Colony Formation assays (Figs. 5A–5C). Additionally, wound healing and Transwell invasion assays demonstrated that CCL20 knockdown markedly suppressed the migratory and invasive capacities of gastric cancer cells compared to controls (Figs. 5D–5G). IHC staining analysis of gastric cancer tissue samples (n = 37) revealed that CCL20 is highly expressed in tumor tissues relative to adjacent normal tissues (Figs. 5H, 5I). Additional analyses using patient clinical information showed a strong correlation between elevated CCL20 expression and lymph node metastasis (Figs. 5J, 5K, and Table S5). No significant associations were observed between CCL20 expression and pathological stage, degree of differentiation, or patient gender (Figs. S3C–S3E). These results indicate that the oncogenic gene CCL20 plays an important role in the biological function of GC cells.

BNC1 acts on the CCL20 promoter to mediate the JAK-STAT signaling pathway to promote apoptosis in gastric cancer cells

KEGG analysis of transcriptome sequencing data revealed significant enrichment of the chemokine signaling pathway in gastric cancer samples (Fig. 4I). Previous studies have shown that CCL20 mediates the JAK-STAT signaling pathway, influencing migration and invasion in breast cancer cells (Muscella, Vetrugno & Marsigliante, 2017), and that berberine inhibits apoptosis in gastric cancer via the JAK-STAT pathway (Xu et al., 2022). In the current study, initial functional assays indicated that overexpression of BNC1 promotes apoptosis in gastric cancer cells (Figs. 2M–2P). These findings suggest that BNC1 acts on the CCL20 promoter to mediate the JAK-STAT signaling pathway, thereby promoting apoptosis. Consistent with this hypothesis, overexpression of BNC1 or knockdown of CCL20 led to decreased expression of JAK2 and inhibited phosphorylation of STAT3 (Figs. 6A–6D). This suppression resulted in diminished expression of the anti-apoptotic protein BCL-2 (Fig. 6E) and an increased expression of the pro-apoptotic protein BAX (Fig. 6F). These results suggest that BNC1 acts on the CCL20 promoter to mediate the molecular mechanism by which JAK-STAT signaling promotes apoptosis.