Regents

Dulbecco's modified Eagle medium (DMEM, 02-5062EJ), fetal bovine serum (FBS, 16140-071), penicillin–streptomycin (Pen–Strep, 15140-122), and Opti-MEM serum-reduced medium (31985-070) were purchased from Gibco (Semitic, NY, USA). LipofectamineTM 2000 (Lipo 2000, 11668-019) was obtained from Invitrogen (Waltham, MA, USA). Puromycin was obtained from Solarbio (Beijing, China). Enhanced chemiluminescence (ECL, BL520B) kit was purchased from Biosharp (Hefei, China). MTT reagent (M2128) and DMSO (D2650) were obtained from Sigma-Aldrich (St. Louis, MO, USA); SYBR (Q311‐02/03) was from Vazyme (Nanjing, China); citrate buffer (G1202), DAB solution (G1212), and hematoxylin (G1004) were from Servicebio (Wuhan, China).

Cells

The two human DLBCL cell lines, SU-DHL-2 (YDT-0623) and U2932 (YDT-0681), were purchased from INDIT (Hangzhou, China). Cells were seeded in a 25 cm2 cell culture flask and cultured in DMEM medium containing 10% FBS and 1% Pen–Strep with 5% CO2 at 37°C.

Transient transfection

All siRNA constructs (siR-LARS, siR-LRPPRC, siR-HIF-1α, and siR-NC) were obtained from Hippo Bio (Huzhou, China), while expression plasmids (pcDNA3.1-LARS, pcDNA3.1-LRPPRC, pcDNA3.1-HIF-1α, and empty pcDNA3.1 vector) were purchased from Youbio (Hunan, China). For transient transfections, SU-DHL-2 and U2932 cells were seeded in six-well plates at 70–80% confluence prior to transfection. siRNA transfections were performed using 100 pmol of siRNA (final concentration 50 nM) complexed with 5 μL of Lipofectamine 2000 in Opti-MEM serum-reduced medium, representing a Lipofectamine:siRNA ratio of 1:1 (v/v), with cells harvested 24 h post-transfection for analysis. Plasmid transfections utilized 4 μg of DNA combined with 10 μL of Lipofectamine 2000 (ratio 2.5:1 (v/w)), followed by 24 h of incubation before assessment. Transfection efficiency was validated by western blot analysis.

Construction of LARS stably overexpressed SU-DHL-2 cells

For the construction of LARS stably overexpressed SU-DHL-2 cells, the LARS-overexpressed lentivirus (Lenti-LARS) and the corresponding control lentivirus (Lenti-control) were purchased from GeneChem (Shanghai, China). When the confluence of SU-DHL-2 cells reached 70%, cells were co-incubated with the Lenti-LARS or Lenti-control (at a titer of 1.0 ×108 Tu·ml-1) for 48 h. Then 1 μg/mL puromycin was utilized for screening.

Animal models

Twelve male BALB/c nude mice (4–5 weeks old) purchased from SLAC Laboratory Animal Co., Ltd (Shanghai, China) were housed under specific pathogen-free (SPF) conditions with controlled temperature (22 ± 1 °C), humidity (50 ± 10%), and a 12-h light/dark cycle. Mice were randomly divided into two groups (n = 6 per group) and subcutaneously injected with SU-DHL-2 cells stably overexpressing LARS or control vector (5×106 cells suspended in 100 μL PBS). Tumor growth was monitored every 3 days by caliper measurements (volume calculated as *V = 0.5 × length × width2*). When tumors reached the humane end point (≥2000 mm3) or showed signs of distress (e.g., >20% weight loss, ulceration), mice were euthanized by cervical dislocation. Excised tumors were measured for volume and weight. The animal experiments were conducted in accordance with the protocols approved by the Medical Ethics Committee of Jiangxi Cancer Hospital (Approval No. 2025ky007). All procedures strictly followed the institutional guidelines for animal welfare and the ARRIVE guidelines.

Western blot (WB)

The western blot procedures were performed as previously described with the following modifications [13]. Total protein was extracted from tumor tissues and SU-DHL-2 and U2932 cells using RIPA lysis buffer supplemented with protease and phosphatase inhibitors. Protein samples (20–30 μg per lane) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (0.45 μm pore size). Membranes were blocked with 5% bovine serum albumin (BSA) in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 h at room temperature to prevent nonspecific binding.

Primary antibodies were diluted in 5% BSA/TBST according to the concentrations listed in Table 1 and incubated with membranes overnight at 4°C. After washing, membranes were probed with species-matched horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit IgG, CST 7074P2, 1:2000; anti-mouse IgG, CST 7076P2, 1:2000) in 5% BSA/TBST for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) kit with exposure times ranging from 1 to 90 s (optimized for each target protein) on a ChemiDoc imaging system. Image analysis was performed using Image Lab software (Bio-Rad).

Table 1 Details of antibodiesMTT assay

The MTT assay was carried out to detect the cell proliferation of SU-DHL-2 and U2932 after transfection treatment. Cells were seeded into a 96-well plate at a density of 2×103 cells/well and cultured for 0, 24, 48, and 72 h. The MTT reagent was diluted to 1 mg/mL with phosphate buffer solution (PBS) and then added to the 96-well plate containing cell suspensions (50 μL per well) for co-incubation for 3 h at 37°C. After the addition of DMSO (150 μL per well) and the dissolution of crystallization, the results were quantified utilizing a microplate reader (Molecular Device, California, USA) at the absorbance of 570 nm.

Flow cytometry

Flow cytometry was applied to determine the apoptosis of SU-DHL-2 and U2932 cells after transfection treatment. The procedures were completed using the Annexin V–FITC/PI Apoptosis Detection Kit (4A Biotech, Beijing, China, FXP018) according to the manufacturer's protocol. Then the results were detected with a flow cytometer.

LA detection assay

The expression of LA was determined using the human LA enzyme-linked immunosorbent assay (ELISA) detection kit (MEIMIAN, Jiangsu, China, 12907) according to the producer’s instructions. Then the OD value of the samples was examined with the microplate reader at the absorbance of 450 nm.

Immunohistochemistry (IHC)

The IHC assay was implemented to detect the expression of HK2 and GLUT1 in the tumor tissues. The tissues cut into sections were de-paraffinized, and then successively soaked in citrate buffer for antigen retrieval and in 3% H2O2 for endogenous peroxidase removal. Subsequently, sections were blocked with 3% BSA and then incubated with antibodies specific for HK2 and GLUT1 overnight. After three washes with PBS, slides were incubated in secondary antibodies. Then the sections were stained with the DAB solution and counterstained with hematoxylin. The results were observed with a microscope.

Bioinformatics

Bulk RNA-seq data (GSE25297) were analyzed using limma in R, with log2(TPM) transformed values compared between DLBCL (n = 7) and normal B cells (n = 7) via t-test. scRNA-seq data (GSE182434) were processed with Seurat v5.2.1: after quality control, 2000 variable genes were used for PCA (30 PCs) and UMAP clustering (resolution = 0.5). B cells (CD19+/MS4A1+) were stratified into the LARS+ (TPM≥1) and LARS− (TPM<1) groups for proportion (Chi-square test) and LRPPRC expression (Wilcoxon test) analyses. All visualizations used ggplot2/ggpubr in R.

The survival curve was generated based on The Cancer Genome Atlas (TGCA) database utilizing GraphPad Prism 6.0. The differentially expressed genes (DEGs) between the LARS high- and low-expression samples were obtained using DESeq2 R Package 1.22.2 and exhibited using the volcano plot. Based on the information of DEGs, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were carried out by Ensembl BioMart101. The protein of LARS-overexpressed SU-DHL-2 cells was extracted and mass spectrometry was performed by LC-BIO (Hangzhou, China) to obtain the differentially expressed proteins (DEPs). Then the results were presented as the heatmap, which was generated by R version 3.6.3.

Dual-luciferase reporter assay

The putative HIF-1α binding site within the HK2 3'UTR region and its mutated variant were amplified and cloned into the PGL3-basic vector (Promega, Madison, WI, USA). Constructs (HK2-3'UTR-WT and HK2-3'UTR-Mut) along with empty PGL3 vector were co-transfected with pRL-TK Renilla luciferase control vector into SU-DHL-2 cells using Lipofectamine 2000. Cells were harvested 24 h post-transfection, and firefly luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) and normalized to Renilla luciferase activity. All transfections were performed in three independent biological replicates (n = 3).

Chromatin immunoprecipitation (ChIP)

The chromatin immunoprecipitation (ChIP) assay was performed using the ChIP-Seq High Sensitivity Kit (Abcam, ab185908) with the following optimized conditions. Briefly, 1×107 SU-DHL-2 or U2932 cells per immunoprecipitation reaction were cross-linked with 1% formaldehyde for 10 min at room temperature, followed by quenching with 125 mM glycine. Chromatin was sheared to 200–500 bp fragments using a Bioruptor Pico sonicator (30 s ON/30 s OFF, 15 cycles).

Immunoprecipitation was performed overnight at 4 °C using 5 μg of anti-HIF-1α antibody (Abcam, ab179483) with protein A/G MagPoly beads. Normal rabbit IgG (Abcam, ab172730) served as negative control. After reverse cross-linking and DNA purification, the enriched DNA fragments were analyzed by qPCR using the following primers specific to the HK2 promoter region:

forward: 5'-TTCCGTCCCAGCCTTTAGCC-3',

reverse: 5'-TCATCGCTCACGGCTCGC-3'.

The PCR amplification was performed with SYBR Green Master Mix under the following conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. All ChIP-qPCR results were normalized to input DNA and expressed as fold enrichment relative to the IgG control.

Statistical analysis

Every experiment was repeated at least three times, and the typical data were selected and analyzed with GraphPad Prism 6.0. t-test was used for comparison between two groups, and one-way ANOVA for that of multiple groups. p<0.05 was regarded as statistically significant.