One gastric epithelial cell line (GES - 1) and three GC cell lines (MKN74, HGC27, AGS, SNU216) were selected from Chinese Academy of Sciences (Shanghai). MKN74 and HGC27 cells were RPMI - 1640 (cat No. CAT#01-100-1ACS, BI) containing 10% FBS. AGS cell was cultured in DMEM/F12 (cat No. CAT#01-172-1ACS, BI) + 10% FBS (cat No. CAT#SA102.02, Cellmax). The culture environment was 37 °C and 5% CO2 concentration. For the exogenous lactate treatment, the L-lactate (Sigma, L1750) was added to each cell group (20 mmol/L). Besides, the carcinoma-associated fibroblasts (CAFs)-conditioned medium (CM) was prepared for exogenous lactate treatment.

For the cell transfection, the lentiviral vectors for stable knockdown and overexpression of HNRNPC were provided by GeneChem (Shanghai, China). GC cells were seeded in 24-well plates, and then for transfection with the concentrated virus (MOI = 100) when cells reached 60%-70%. Then, the infected cells were treated with puromycin (Sigma-Aldrich, 2 μg/mL).

Total RNA was prepared from cells using TRIzol (Tiangen, cat No. DP430) according to its instructions of manufacturer. The quality of RNA samples was evaluated via spectrophotometric analysis and then subjected to HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Shanghai, China) to generate cDNA. ChamQ SYBR qPCR Master Mix (Vazyme, Q311 - 02/03) was used for real-time PCR to determine the mRNA level relative to beta-actin by the 2^−ΔΔCt method. The sequences of primers used in this study were shown in Supplementary Table S1 .

Single-cell suspensions were prepared from fresh tissues using enzymatic digestion and mechanical dissociation. Cell viability (>85%) and concentration were confirmed via trypan blue staining and automated counting. Approximately 10,000 cells per sample were loaded onto a 10x Genomics Chromium Controller to generate single-cell Gel Bead-In-EMulsions (GEMs). Libraries were constructed using the Chromium Single Cell 3′ Reagent Kit v3.1, followed by paired-end sequencing (150 bp) on an Illumina NovaSeq 6000 platform at a depth of ~50,000 reads per cell. Raw sequencing data were processed via Cell Ranger (v7.1.0) for alignment, filtering, and UMI counting. Downstream analyses, including normalization, dimensionality reduction (PCA, UMAP), and cluster identification, were performed using Seurat (v5.0.1) and Scanpy (v1.9.3). Low-quality cells (mitochondrial genes >20% or UMIs <500) were excluded. Cell types were annotated via marker gene expression from public databases.

The proliferation of GC cells was tested by EdU and CCK - 8 assay. In brief, the transfected GC cells were utilized. EdU incorporation assay was carried out with an EdU kit (Roche, Mannheim, Germany). The oxaliplatin resistance was tested using CCK - 8 for the IC₅₀ value (50% maximal inhibitory concentration).

For lactate production analysis, GC cells were cultured with a completed medium (24 h) and the culture medium was changed by fresh medium. After incubation (6 h) of cultured medium, the lactate content was detected by stable isotope tracing analysis (18) or L-Lactate Assay Kit (Colorimetric, ab65331, Abcam).

ECAR was determined using Seahorse XF96 Analyzer Glycolysis kit (Agilent Technologies, Cat No. 103344) and the OCR was detected using Seahorse XF Cell Mito Stress Test kit (Cat No. 103015). GC cells (10⁴) were inoculated into XF96 culture plates. Then, ECAR was determined after saturating concentration of glucose, oligomycin and 2-deoxyglucose (2-DG) addition at the indicated time points. OCR was determined by adding oligomycin, Trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) and Rotenone/antimycin A treatment. ECAR (mpH/min) and OCR (pmol/min) were automatically calculated via Seahorse XFp software.

The iron concentration was detected using the Cell Total Iron Colorimetric Assay Kit (Cat. E-BC-K880-M, Elabscience) according to the manufacturer’s instructions. The GSH was tested by commercialized GSH/GSSG assay kit (Beyotime, Cat. S0053). The lipid ROS level was tested by BODIPY™ 581/591 C11 reagent (Cat. D3861, Invitrogen, California, USA).

GC cells were embedded and stained for the examining mitochondrial morphology. The sample images were observed under a transmission electron microscope (HT7700, HITACHI).

To confirm molecular interactions, RIP analysis was performed using a RNA Binding Protein Immunoprecipitation Kit (Magna, Shanghai, China). A total of approximately 10⁷ GC cells were collected and lysed. After removal of DNA, lysates were incubated with RIP buffer containing anti-HNRNPC antibody (Abcam, 1:1000, Cat No. ab314004) and normal control IgG (Bioss, Cat No. bs-0295PC) were incubated for 16 hours at 4°C. The RNA-protein complexes were then incubated with protein A/G balanced magnetic beads. After elution of RNA, the immunoprecipitated RNA was extracted for the A/G magnetic beads, and qRT-PCR was performed.

Transfected GC cells were treated with 8 μg/ml actinomycin D (Act D) for 0, 3, 6, and 9 hours, respectively, and RNA was extracted for reverse transcription and qRT-PCR. Relative quantitation was calculated using the 2^-ΔΔCt method and normalized to β-actin. Calculation of the half-life of MCT1 mRNA was conducted.

A subcutaneous transplanted tumor model was established by subcutaneously injecting 100 μL of MKN74 cells suspension into the flank of BABL/c nude mice that housed in specific pathogen-free (SPF) animal facility. Tumor size was measured once three days, and the calculation formula was: volume = length×width²/2. Three weeks after injection, tumors were harvested, weighed, and stored for further study. All animal experiments were conducted in strict compliance with the National Institutes of Health Ethical Principles and Guidelines for the Care and Use of Animals. This study had been approved by the Committee on Animal Research of Zibo Central Hospital.

Statistical analysis was performed using GraphPad Prism 8.0 software and Student’s t test or ANOVA (multiple comparisons between multiple groups). Unless otherwise stated, data are expressed as mean ± standard deviation. Kaplan-Meier method and log-rank test were used for overall survival analysis. The p-value is 0.05, and the difference is statistically significant.

To investigate whether HNRNPC affects the GC progression, the HNRNPC expression was tested in multitudinous human cancers, and results illustrated that HNRNPC level significantly elevated, especially the GC ( Figure 1A ). The HNRNPC was a remarkable high-expression one as comparing to normal control group ( Figure 1B ). In GC cells, the HNRNPC level was found to be highly expressed ( Figure 1C ). Besides, the HNRNPC was also tested in the scRNA-Seq ( Figure 1D ). Results indicated that HNRNPC and SLC16A1 (MCT1) were both up-regulated in the GC samples ( Figure 1E ). Conclusively, the results obtained from this study affirmed the elevated HNRNPC expression in GC.

To explore the function of HNRNPC on GC malignant phenotypes, the series of assays were performed. Firstly, the proliferation assay indicated that HNRNPC silencing repressed the proliferation and HNRNPC overexpression promoted it ( Figure 2A ). In our analysis, our data indicated that HNRNPC over/silencing was closely correlated to the lactate generation in the GC cells, thus the following assays was performed to investigate the lactate-related index. The lactate accumulation in the culture environment was detected and results indicated that HNRNPC silencing repressed the lactate quantity, and HNRNPC overexpression promoted the lactate ( Figure 2B ). Moreover, the HNRNPC silencing increased the pH value of the culturing medium, and HNRNPC overexpression reduced the pH value of the culturing medium ( Figure 2C ). To confirm the effect of HNRNPC, ECAR and oxygen consumption rate (OCR) were detected. ECAR of seahorse glycolytic stress tests demonstrated that the activation of glycolysis in GC cells was significantly reduced by HNRNPC silencing, in contrast to that of HNRNPC overexpression ( Figures 2D, E ). OCR of oxygen consumption rate results demonstrated that HNRNPC silencing enhanced mitochondrial respiration in GC cells, while HNRNPC overexpression reduced the mitochondrial respiration ( Figures 2F, G ). Conclusively, the results obtained from this study affirmed that HNRNPC positively fortified the aerobic glycolysis and lactate accumulation in GC.

Given that previous findings showed the role of HNRNPC silencing on lactate inhibition, the further assays were performed to verify the interaction within HNRNPC and lactate in GC. The exogenous lactate was administrated to the culture to stimulate the GC cells, including lactate (L- lactate) and conditioned medium (CM). Carcinoma-associated fibroblasts (CAFs) is considered as one of the most abundant components in tumor and the major lactate source in the extracellular environment (19). CAFs-CM with elevated lactate level was prepared for culture of GC cells. The following assays monitored the GC cells’ response to HNRNPC/lactate treatment. Proliferation assay indicated that lactate and CM both recovered the inhibition by HNRNPC silencing ( Figure 3A ). The oxaliplatin chemotherapy sensitivity test showed that lactate and CM both promoted the half maximal inhibitory concentration (IC₅₀) of GC cells toward oxaliplatin ( Figure 3B ). Thus, exogenous lactate could accelerate the oxaliplatin resistance of GC cells. ECAR analysis revealed that lactate and CM both promoted the glycolytic capacity and glycolytic rate of GC cells with HNRNPC silencing ( Figures 3C, D ). Then, the OCR analysis unveiled that lactate and CM both reduced the OCR at both basal and maximal respiratory rate levels of GC cells with HNRNPC silencing ( Figures 3E, F ). Conclusively, the results obtained from this study affirmed that exogenous lactate accelerated the proliferation, oxaliplatin resistance and aerobic glycolysis in GC that inhibited by HNRNPC silencing.

Emerging literature are revealing the important role of iron-dependent cell death, also known as ferroptosis. Here, this study tried to investigate the role of HNRNPC/Lactate on iron and its relative ferroptosis. Iron deposition is related to intracellular accumulation of iron ions and mitochondrial metabolism. The lactate and CM administration could reduce the iron concentration accumulation, which was also consistent with the ferroptosis specific inhibitor Ferrostatin-1 (Fer-1) ( Figure 4A ). Moreover, HNRNPC silencing up-regulated the iron concentration accumulation, and the lactate, CM and Fer-1 co-administration reduced the iron concentration ( Figure 4B ). Moreover, HNRNPC silencing aggravated the ROS level ( Figures 4C, D ), reduced the GSH ( Figure 4E ) and exacerbated the mitochondrial injury ( Figure 4F ). Conclusively, the results affirmed that lactate accelerated the ferroptosis resistance in GC with HNRNPC silencing.

The previous results indicated that HNRNPC regulated the lactate accumulation in GC cells, thus the following assays were performed to explore the mechanism. The RNA-Seq in the sh-NC and sh-HNR-1# group was tested ( Figure 5A ). Gene set enrichment analysis (GSEA) of the RNA-Seq revealed that monocarboxylic acid transport was altered ( Figure 5B ). In the m⁶A sequencing data, the m⁶A modified profile was detected ( Figure 5C ). In the seq-data, the visualization tools showed that there was vital m⁶A modified site on the 3’-UTR of MCT1 gene ( Figure 5D ). Given the vital function of HNRNPC on aerobic glycolysis and lactate accumulation in GC, the following assays were performed to verify which element participated in the progression. Results indicated that MCT1 exerted the more remarkable alteration upon HNRNPC overexpression ( Figure 5E ). In the public database, the correlation analysis revealed that HNRNPC expression was positively correlated to the MCT1 (SLC16A1 gene) level in STAD (Stomach adenocarcinoma) samples ( Figure 5F ). RIP-PCR assay was performed and data revealed that HNRNPC significantly interacted with the MCT1 mRNA in GC cells ( Figure 5G ). Then, the RIP-PCR assay also illustrated that lactate and CM both increased the enrichment of MCT1 mRNA in incorporation within MCT1 mRNA and anti-HNRNPC ( Figure 5H ). The immunoprecipitation data revealed that exogenous lactate administration could enhance the binding within HNRNPC. For the mRNA fata of MCT1, the RNA decay assay illustrated that HNRNPC silencing reduced the MCT1 mRNA stability (half life time, t_1/2), and the lactate and CM both increased the MCT1 mRNA stability ( Figure 5I ). Conclusively, the results obtained from this study affirmed that MCT1 was identified as the downstream target of HNRNPC, which was regulated by the lactate microenvironment.

Our previous findings showed that HNRNPC regulated the aerobic glycolysis and lactate accumulation and ferroptosis in GC. Besides, MCT1 was identified as the downstream target of HNRNPC. Thus, to verify the function of HNRNPC/MCT1 on the GC malignant phenotype, the rescue assays were performed in this part. The iron concentration, pH value, ECAR and OCR were respectively detected. MCT1 special inhibitor AZD3965 and MCT1 small interfering RNA (siRNA) up-regulated the iron concentration, increased the pH value and reduced the glycolysis ( Figures 6A–D ). Exogenous lactate and ferroptosis specific inhibitor Ferrostatin-1 (Fer-1) reduced these indexes that up-regulated by si-MCT1. These assays confirmed the role of HNRNPC, MCT1 and lactate on the GC aerobic glycolysis and ferroptosis resistance. Therefore, HNRNPC targeted MCT1 to fortify the aerobic glycolysis and lactate accumulation in GC, thereby accelerating the ferroptosis resistance.

To explore whether HNRNPC influenced the tumor growth of GC in vivo, the xenograft mice assay was performed using nude mice ( Figure 7A ). In the tissue, the tumor volume and weight were reduced in the HNRNPC silenced GC cells inoculation ( Figures 7B, C ). Metastatic progression by noninvasive bioluminescence In Vivo Imaging System revealed that HNRNPC silencing reduced the tumor growth ( Figure 7D ). Besides, mIHC revealed that the MCT1 protein was also reduced in the HNRNPC silenced group ( Figure 7E ). Besides, the MCT1 level was also decreased in HNRNPC silenced GC cells inoculation ( Figure 7F ). Overall, these data indicated that HNRNPC silencing repressed the tumor growth of GC.