The ‘Expression Boxplot’, ‘Expression Stage Plot’ and ‘Survival Plots’ modules on the GEPIA database (http://gepia.cancer-pku.cn/index.html) were used to analyze the expression of OLR1 in tumor and adjacent tissues and its correlation with tumor stage and survival in thyroid carcinoma.

A total of 20 PTC patients (between April 2024 and July 2025) were included, with median age of 46.5 years (range: 29–58 years). Among them 3 (15%) were males and 17 (85%) were females. Paired tumor and adjacent tumor tissues (~1 cm from the tumor margin) were obtained both retrospectively using stored samples and prospectively. The tissues were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin and cut into 4-µm slices. The slides were deparaffinized, rehydrated and antigen-restored, successively. After removing the endogenous peroxidase in 3% H₂O₂, the slices were blocked by 5% goat serum (Wuhan Servicebio Technology Co., Ltd.) for 30 minutes (min) at room temperature and cultivated in anti-LOX-1 antibody (1:200; cat. no. DF6522, Affinity Biosciences) or anti-H3K18la (1:500; cat. no. PTM-1406RM, PTM BIO) at 4°C overnight. Subsequently, the slices were incubated with anti-rabbit-HRP secondary antibody (1:2,000; cat. no. GB23303, Wuhan Servicebio Technology Co., Ltd.) at room temperature for 30 min. Finally, a DAB kit (Wuhan Servicebio Technology Co., Ltd.) was used to develop the color. The slices were counterstained by hematoxylin (Wuhan Servicebio Technology Co., Ltd.) at room temperature for 3 min. The images were captured under an inverted microscope (Motic Incorporation, Ltd.). The slices were analyzed by two pathologists, independently. The IHC score was the result of the staining intensity (score of 0, 1, 2 or 3) multiplied by the staining percentage (score of 1, 2, 3 or 4), of which the highest was 12. The final score was the average assessment by two pathologists. The present study was approved by the Ethics Committee of Harbin Medical University Cancer Hospital with approval number YD2024-13 on November 4, 2024. The Ethics Committee formally waived the requirement for additional informed consent for the study. Moreover, participants had provided written consent for the secondary use of biological specimens and medical data as part of routine clinical procedures at our institution.

The human PTC cell lines TPC-1 (Cellverse Bioscience Technology Co., Ltd.) and IHH-4 (Cellverse Bioscience Technology Co., Ltd.) were obtained. RPMI-1640 (Procell Life Science & Technology Co., Ltd.) with 10% fetal bovine serum (Procell Life Science & Technology Co., Ltd.) was used to culture the cells in 5% CO₂ and at 37°C.

Negative control (NC) overexpression plasmid (oeNC; 0.8 µg; Shanghai GenePharma Co., Ltd.), OLR1 overexpression plasmid (oeOLR1; 0.8 µg; Shanghai GenePharma Co., Ltd.), NC siRNA (siNC; 50 nM; Shanghai GenePharma Co., Ltd.) and OLR1 siRNA (siOLR1; 50 nM; Shanghai GenePharma Co., Ltd.) were transfected into TPC-1 and IHH-4 cells at 37°C for 6 h. Transfect-mate (Shanghai GenePharma Co., Ltd.) was applied to complete the transfection. At 48 h after transfection, LOX-1 (OLR1 coding protein) expression, LDHA expression, cell proliferation, cell apoptosis, cell invasion, lactate concentration, H3K18la expression and LEM domain containing 1 (LEMD1) expression were assessed. The siRNA sequences were: siOLR1 (sense, 5′-CAGCUGAUCUGGACUUCAUTT-3′, antisense, 5′-AUGAAGUCCAGAUCAGCUGTT-3′) and siNC (sense, 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense, 5′-ACGUGACACGUUCGGAGAATT-3′).

The TPC-1 and IHH-4 cells were incubated with 10 mM L-sodium lactate (Shanghai Aladdin Biochemical Technology Co., Ltd.) and 10 mM sodium oxamate (Shanghai Aladdin Biochemical Technology Co., Ltd.), respectively. H3K18la expression and H3K18la enrich level on the LEMD1 promoter were evaluated.

oeNC and oeOLR1 were transfected into TPC-1 and IHH-4 cells. Meanwhile, 10 mM sodium oxamate was added and incubated with the cells. Afterwards, H3K18la expression, LEMD1 expression, cell proliferation, cell apoptosis and cell invasion were measured.

oeNC (0.8 µg) + siNC (50 nM), oeOLR1 (0.8 µg) + siNC (50 nM), oeNC (0.8 µg) + LEMD1 siRNA (siLEMD1; 50 nM; Shanghai GenePharma Co., Ltd.) and oeOLR1 + siLEMD1 were transfected into TPC-1 and IHH-4 cells in the presence of Transfect-mate (Shanghai GenePharma Co., Ltd.) at 37°C for 6 h. LOX-1 (OLR1 coding protein) expression, LEMD1 expression, cell proliferation, cell apoptosis and cell invasion were analyzed at 48 h after transfection. The siRNA sequences of siLEMD1 were listed: siLEMD1 (sense, 5′-GUGUCUGAGUGACUGUAAATT-3′, antisense, 5′-UUUACAGUCACUCAGACACTT-3′).

Cell counting kit-8 reagent (Wuhan Servicebio Technology Co., Ltd.) was incubated with the cells at 0, 24, 48 and 72 h after transfection for 2 h. The OD value was read at 450 nm and a cell apoptosis kit (Wuhan Servicebio Technology Co., Ltd.) was used to analyze cell apoptosis 48 h after treatment. The cells were incubated in Annexin V and propidium iodide working solution for 15 min in darkness at room temperature. A BD FACSCalibur flow cytometer (BD Biosciences) was used to analyze the cells. The data was analyzed by FlowJo X (BD Biosciences). The percentage of early + late apoptotic cells was calculated. The concentration of lactate was analyzed by a Lactic Acid Content Assay Kit (Abbkine Scientific Co., Ltd.) according to the manufacturer's instructions 48 h after transfection.

After transfection for 48 h, the cells were harvested. Transwell inserts (0.8 µm; Wuhan NEST Biotechnology Co., Ltd.) were cultured with Matrigel Matrix (BD Biosciences) at 37°C for 1 h. After which, the cells (2×10⁴) in phosphate buffer saline were loaded into the upper chamber of Transwell inserts and the lower chamber was filled with 10% FBS-containing RPMI-1640. The inserts were incubated for 24 h at 37°C. Afterwards, the cells were stained in crystal violet solution (Wuhan Servicebio Technology Co., Ltd.) at room temperature for 5 min after being incubated with 4% paraformaldehyde at room temperature for 15 min. Finally, the invasive cells were counted with a microscope (Motic Incorporation, Ltd.).

The cells were lysed to extract total protein using RIPA buffer (Wuhan Servicebio Technology Co., Ltd.) 48 h after transfection or lactate/oxamate incubation. Total protein was quantified using a BCA kit (Wuhan Servicebio Technology Co., Ltd.). A total of 10 µg thermally denatured protein was loaded on 4–20% pre-cast gels, separated and transferred to a nitrocellulose membrane (Whatman plc; Cytiva). The membrane was then blocked in 5% skimmed milk (Wuhan Servicebio Technology Co., Ltd.) at 37°C for 60 min. Primary antibodies, including anti-LOX-1 antibody (1:2,000; cat. no. DF6522, Affinity Biosciences), anti-LEMD1 antibody (1:1,000; cat. no. CSB-PA715035LA01HU, Cusabio Technology, LLC) anti-H3K18la (1:2,000; cat. no. PTM-1406RM, PTM BIO), anti-H3 antibody (1:5,000; cat. no. AF0863, Affinity Biosciences), LDHA antibody (1:2,000; cat. no. DF6280, Affinity Biosciences) and anti-GAPDH antibody (1:3,000; cat. no. GB15004, Wuhan Servicebio Technology Co., Ltd.), were cultivated with the membrane overnight at 4°C. After which, anti-rabbit-HRP secondary antibody (1:20,000; cat. no. GB23303, Wuhan Servicebio Technology Co., Ltd.) was incubated with the membrane for 90 min. Finally, the protein bands were illuminated with an ECL kit (MeilunBio). The western blotting images were quantified using ImageJ software (v1.8; National Institutes of Health).

The cells (1×10⁷) were crosslinked with formaldehyde for 10 min at 37°C and suspended in sodium dodecyl sulfate buffer. After which, the cells were ultrasonically lysed (25% frequency; 30 sec on, 30 sec off, 20 cycles) on ice and centrifuged. The lysate (100 µl) was incubated with IgG (1:50) or H3K18la (1:50) antibody at 4°C overnight, followed by incubation with protein A+G Agarose (Beyotime Biotechnology) for 1 h, and the 1 µl lysate was used as input. After incubation, the lysate was centrifuged (1,000 × g) at 4°C for 10 min and washed with specific buffer (IP Elution Buffer), successively. After which, the precipitate was reverse crosslinked and the DNA was analyzed with Taq DNA Polymerase containing-SYBR Green qPCR Master Mix (Wuhan Servicebio Technology Co., Ltd.). The thermal cycle procedures were as follows: 95°C for 2 min, 1 cycle; 95°C for 15 sec, 61°C for 30 sec, for 40 cycles. The data was calculated using the %Input method.

The GV492 plasmid (Shanghai Genechem Co., Ltd.) and GV872 plasmid (Shanghai Genechem Co., Ltd.) were used to establish OLR1 overexpression plasmid and LEMD1 knockdown plasmid, respectively. The lentivirus was constructed through second generation system. The 20 µg OLR1 overexpression or LEMD1 knockdown plasmid, 15 µg pHelper 1.0 plasmid and 10 µg pHelper 2.0 plasmid were transfected into 293T cells (Cellverse Bioscience Technology Co., Ltd) at 37°C for 6 h. The supernatant was collected to harvest lentivirus at 48 h after transfection. The TPC-1 cells were infected with OLR1 overexpression lentivirus, LEMD1 knock-down lentivirus alone or in combination (Shanghai Genechem Co., Ltd.). The multiplicity of infection (MOI) was 20. Moreover, control lentivirus (Shanghai Genechem Co., Ltd.) was infected the TPC-1 cells. The stably infected TPC-1 cells were selected with 1.5 µg/ml puromycin for 7 days. BALB/c nude mice with 4–6 weeks old and 18–22 g (Shanghai SLAC Laboratory Animal Co, Ltd.) were housed in specific pathogen-free facility and randomly assigned into four groups (three mice per group). The stably infected TPC-1 cells were inoculated on the right flank of the mice (4×10⁶ cells per mouse). A caliper was used to measure tumor volume every week. The mice were sacrificed by cervical dislocation at four weeks. The tumors were harvested, weighted, fixed and cut into 4 µm sections. The IHC was performed as aforementioned, with the application of anti-LOX-1 antibody (1:200), anti-H3K18la antibody (1:500) and anti-LEMD1 antibody (1:100). The animal experiments were approved by the Ethics Committee of Harbin Medical University Cancer Hospital with approval number YD2024-13 and Wuhan Servicebio Institutional Animal Care and Use Committee (IACUC) with approval number 2025079-2.

All data in the present study were analyzed by GraphPad 9.0 software (Dotmatics). The experiments were performed in triplicate (n=3 in each group). Prior to conducting parametric tests, the normality and homogeneity of variance were analyzed by Shapiro-Wilk test and Brown-Forsythe test, respectively. If these assumptions were met, one-way analysis of variance with Tukey's or Dunnett's test was used for comparison among groups. A paired t-test was applied for comparison between tumor and paired-adjacent tumor groups. Pearson correlation coefficient test was adopted to analyze the correlation of LOX-1 and H3K18la expression. P<0.05 was considered to indicate a statistically significant difference.

Referring to the analyses based on the GEPIA public database, OLR1 expression was upregulated in thyroid cancer tissues compared with non-tumor tissues (Fig. 1A) and its expression differed among patients with thyroid cancer at different TNM stages (Fig. 1B). Moreover, GEPIA also revealed that patients with thyroid cancer with high OLR1 expression presented a worse OS compared with those with low OLR1 expression (Fig. 1C). LOX-1 expression (OLR1 coding protein) was detected by IHC assay in 20 pairs of PTC tumor and adjacent non-tumor tissues for verification, which verified that LOX-1 expression was higher in PTC tumor tissues compared with adjacent non-tumor tissues (Fig. 1D). H3K18la expression was also detected by IHC assay and it showed a higher expression in PTC tumor tissues compared with adjacent non-tumor tissues (Fig. 1E). Moreover, LOX-1 expression was positively associated with H3K18la expression in PTC tumor tissues (Fig. 1F). LOX-1 expression was also positively associated with tumor size of PTC (Fig. S1).

LOX-1 expression was upregulated after oeOLR1 transfection, but downregulated after siOLR1 transfection in TPC-1 and IHH-4 cells (Fig. 2A and B), indicating the success of transfection. After which, cell proliferation, apoptosis and invasion were detected after transfection. oeOLR1 promoted cell proliferation, while siOLR1 inhibited cell proliferation in TPC-1 and IHH-4 cells (Fig. 2C). By contrast, oeOLR1 repressed the cell apoptosis rate but siOLR1 enhanced the cell apoptosis rate in TPC-1 and IHH-4 cells (Fig. 2D and E). Moreover, oeOLR1 increased the invasive cell count while siOLR1 reduced the invasive cell count in TPC-1 and IHH-4 cells (Fig. 2F and G).

oeOLR1 accelerated the production of lactate in TPC-1 and IHH-4 cells, but siOLR1 weakened it (Fig. S2). oeOLR1 also upregulated while siOLR1 downregulated LDHA expression in TPC-1 and IHH-4 cells (Fig. S3A and B). Moreover, oeOLR1 upregulated H3K18la expression, while siOLR1 downregulated H3K18la expression in TPC-1 and IHH-4 cells (Fig. 3A and B). Notably, a previous study reported that H3K18la is directly bound to the promoter of LEMD1 (29). Another study confirmed the oncogene role of LEMD1 in PTC (30). Therefore, the expression of LEMD1 and its relation to H3K18la was subsequently explored in the present study. oeOLR1 increased LEMD1 expression but siOLR1 decreased LEMD1 expression in TPC-1 and IHH-4 cells (Fig. 3A and C). In addition, oxamate reduced H3K18la expression, while lactate elevated H3K18la expression in TPC-1 and IHH-4 cells (Fig. 3D and E). Finally, the ChIP assay verified that H3K18la is bound to the LEMD1 promoter in TPC-1 and IHH-4 cells (Fig. 3F).

Oxamate was used to reduce lactate production along with plasmid transfection. Oxamate lowered H3K18la expression and attenuated the effect of oeOLR1 on H3K18la expression in TPC-1 and IHH-4 cells (Fig. 4A and B). In addition, oxamate downregulated LEMD1 expression and weakened the effect of oeOLR1 on LEMD1 expression in TPC-1 and IHH-4 cells (Fig. 4A and C). Afterwards, oxamate decreased cell proliferation and increased the cell apoptosis rate in TPC-1 and IHH-4 cells, which also attenuated the effect of oeOLR1 on cell proliferation and apoptosis (Fig. 4D-F). Moreover, oxamate reduced the invasive cell count and attenuated the effect of oeOLR1 on the invasive cell count in TPC-1 and IHH-4 cells (Fig. 5A and B).

LEMD1 expression was reduced and the effect of oeOLR1 on LEMD1 expression was weakened by siLEMD1 transfection in TPC-1 and IHH-4 cells. However, LOX-1 expression was not affected by siLEMD1 transfection (Fig. 6A-C). siLEMD1 decreased cell proliferation but increased the cell apoptosis rate in TPC-1 and IHH-4 cells, which attenuated the effect of oeOLR1 on these functions (Fig. 6D-F). Furthermore, siLEMD1 repressed the invasive cell count and weakened the effect of oeOLR1 on the invasive cell count in TPC-1 and IHH-4 cells (Fig. 7A and B).

oeOLR1 elevated tumor volume during observational period, maximum tumor volume and maximum tumor diameter (Fig. 8A and B). Moreover, oeOLR1 increased tumor weight (Fig. 8C) and upregulated the expressions of LEMD1 and H3K18la (Fig. 8D). By contrast, shLEMD1 reduced tumor volume during observational period, maximum tumor volume, maximum tumor diameter and weight, which also attenuated the effect of oeOLR1 in xenograft mice, but it did not affect the expressions of LOX-1 (OLR1 coding protein) and H3K18la. Moreover, the LOX-1 expression was upregulated in oeOLR1 group compared to Control group, indicating oeOLR1 transfection success; the LEMD1 expression was downregulated in shLEMD1 group compared with Control group, indicating shLEMD1 transfection success (Fig. 8D). Regarding the detailed values of maximum tumor volume and diameter: the maximum tumor volumes were 561±117 mm³, 1136±171 mm³, 241±18 mm³, 674±45 mm³ in the Control, oeOLR1, shLEMD1 and oeOLR1+shLEMD1 groups, respectively; and the maximum tumor diameters were 13.2±0.3 mm, 16.2±1.1 mm, 9.2±0.9 mm and 13.5±1.1 mm in the Control, oeOLR1, shLEMD1 and oeOLR1+shLEMD1 groups, respectively.