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TET2-mediated 5-hydroxymethylcytosine of TXNIP promotes cell cycle arrest in systemic anaplastic large cell lymphoma

Clinical Epigenetics volume 17, Article number: 10 (2025) Cite this article

Abstract

Background

5-Hydroxymethylcytosine (5hmC) modification represents a significant epigenetic modification within DNA, playing a pivotal role in a range of biological processes associated with various types of cancer. The role of 5hmC in systemic anaplastic large cell lymphoma (ALCL) has not been thoroughly investigated. This study aims to examine the function of 5hmC in the advancement of ALCL.

Methods

Formalin-fixed, paraffin-embedded (FFPE) tumor tissues (n = 46) were obtained from ALCL patients. GEO dataset was used to analyze the expression 5hmC-relative enzymes. Immunohistochemistry was conducted to assess the level of 5hmC and Ten-eleven translocation 2 (TET2) on FFPE samples. The ALK-positive cell line, Su-DHL-1, and the ALK-negative cell line, DL-40, were utilized as in vitro experimental models. RNA-sequencing and hMeDIP-sequencing assays were performed to explore the potential functions of TET2 in cell cycle regulation.

Results

Our study identified a reduction of 5hmC levels in patients with ALCL, which exhibited a positive correlation with TET2 expression. Downregulation TET2 resulted in decreased 5hmC levels and facilitated the progression of the cell cycle in ALCL cell lines. hMeDIP-seq and subsequent functional analyses demonstrated the involvement of thioredoxin interacting protein (TXNIP) in the regulation of ALCL cells. Further mechanistic studies revealed that 5hmC levels influenced TXNIP expression.

Conclusions

Our study underscores the pivotal roles of 5hmC and TET2 in the regulation of cell cycle progression in ALCL. Therapeutic strategies aimed at targeting 5hmC modification or TET2 may offer a novel approach for the management of ALCL.

Background

Anaplastic large cell lymphoma (ALCL) is a subtype of non-Hodgkin’s T cell lymphoma, distinguished by the presence of large, atypical lymphoid cells that express the CD30 antigen. ALCLs account for approximately 2% to 3% of all malignant lymphomas [1, 2]. In clinical practice, ALK + ALCL generally demonstrates a favorable prognosis. However, ALK- ALCL presents a more heterogeneous prognostic outlook, with poor prognosis associated with recurrence and treatment resistance posing major challenges [3, 4]. Thus, elucidating the underlying mechanisms of lymphomagenesis in ALCL holds significant potential for improving both diagnosis and treatment.

5-hydroxymethylcytosine (5hmC) is an important epigenetic modification derived from the oxidation of 5-methylcytosine (5mC) and plays a critical role in biological processes such as gene regulation and cellular differentiation. The presence of 5hmC in the genome is correlated with active transcription and is enriched in gene bodies as well as transcriptional start sites, indicating its role in transcriptional regulation [5, 6]. The ten-eleven translocation (TET) family of enzymes, which includes TET1, TET2, and TET3, is essential for the active demethylation process [7]. The presence of 2-hydroxyglutarate (2-HG) disrupts this process by inhibiting TET activity, leading to reduced 5hmC levels and increased DNA methylation, both of which are associated with tumor suppressor gene silencing and oncogenic pathway activation [8]. Isocitrate dehydrogenase (IDH) enzymes, particularly IDH1 and IDH2, play a significant role in the modification of 5hmC through their involvement in the metabolism of α-ketoglutarate (α-KG). Mutations in these enzymes are commonly observed in various cancers and result in altered metabolic pathways that impact epigenetic modifications [9].

TET enzymes and IDH enzymes play a significant role in 5hmC modification. However, mutations in these enzymes are frequently observed in T cell lymphomas [10,11,12]. Research indicates that global levels of 5hmC are frequently reduced in these malignancies. For instance, in extranodal NK/T cell lymphoma (ENKTL), TET2 has been implicated in disease progression due to its effects on DNA methylation patterns. In ENKTL cell lines, TET2 knockdown resulted in increased 5mC levels and decreased 5hmC, which correlated with enhanced tumor cell proliferation and reduced apoptosis [13]. Moreover, the loss of 5hmC has been widely identified as a frequent event in peripheral T cell lymphomas, further underscoring its significance in T cell malignancies [14].

While previous research highlights the importance of 5hmC in the pathogenesis of T cell lymphomas, its role has only been studied in a limited number of subtypes. There is a notable paucity of research specifically examining its role in ALCL. Therefore, this study seeks to elucidate the involvement of 5hmC modification in the pathogenesis and/or progression of ALCL.

Methods

Patients and samples

Formalin-fixed, paraffin-embedded (FFPE) samples from 46 patients diagnosed with systemic anaplastic large cell lymphoma (ALCL) between June 2010 and December 2022 were utilized in our experiments. The diagnoses were established based on histopathological and immunohistochemical (IHC) criteria, following the guidelines outlined in the 2016 revision of the World Health Organization (WHO) Classification of Tumors of Hematopoietic and Lymphoid Tissues (fourth edition). Clinical data, including age, sex, B symptoms, Ann Arbor stage, treatment, curative effect and follow-up data, were obtained from the electronic medical records (Table S1). This study was approved by the Ethics Committee of Fujian Cancer Hospital.

Immunohistochemistry (IHC) staining

FFPE tissue Sects. (3 μm) were prepared to perform IHC staining. The antibodies used are presented in Table S3. The samples were analyzed using IHC staining scores based on staining ratio and staining intensity in tumor cells or reactive T cells by three independent observers. The staining intensity was graded as follows: no staining (0), weak staining (1), moderate staining (2), strong staining (3). The staining ratio was score as follows: < 5% (0), 5–30% (1), 31–70% (2), and 71–100% (3).The final score was calculated by the following formula: IHC score = intensity score × ratio score. A final score of ≤ 2 was defined as negative staining, and a final score of > 2 was defined as positive staining.

Cell culture and treatment

The ALK + ALCL cell line Su-DHL-1 was purchased from the American Type Culture Collection (ATCC). The ALK- ALCL cell line DL-40 was obtained from Japanese Cancer Research Resources Bank (JRBC). The cells were cultured in pre-configured RPMI 1640 medium enriched with 10% fetal bovine serum, 1% penicillin–streptomycin, and 1% glutamine (all sourced from Gibco). The cultivation was conducted under controlled conditions of 37 °C and 5% CO2. All cell lines were authenticated by short tandem repeat analysis and tested for mycoplasma contamination. For L-Ascorbic acid (L-AA) treatment analysis, the cells were treated with 200 μM L-AA (MCE) for 24 h. For Bobcat339 treatment analysis, the cells were treated with 7 μM Bobcat339 (MCE) for 24 h. For Pifithrin-α (PFTα) treatment analysis, the cells were treated with 20 μM PFTα (MCE) for 8 h.

T cell isolation

Peripheral blood mononuclear cells (PBMC) from healthy donors were isolated by density-gradient centrifugation using Ficoll (Solarbio). CD3 + T cells were obtained from PBMCs using human CD3 T cell sorting magnetic beads (Miltenyi Biotec) in accordance with the manufacturer’s protocol.

RNA extraction and real-time qPCR

Total RNA from cells was extracted using SteadyPure Rapid RNA Extraction Kit (Accurate Biotechnology). cDNA was synthesized by HiScript II Q RT SuperMix (Vazyme) following the manufacturers instruction. Real-time qPCR was performed using SYBR qPCR Master Mix (Vazyme). Relative normalized expression was calculated by the ‘’2 − ΔΔCT’’ method. The sequences of the primers used in this study were designed by the NCBI Primer-Blast (NCBI Web site) or Oligo7 software. Primers are listed in Table S2.

Western blot analysis

Total protein was extracted using RIPA lysis buffer (Beyotime). After denatured, the proteins were subjected to SDS-PAGE gel electrophoresis. Then, the proteins were transferred into a PVDF membrane. After that, the membranes were blocked with 5% skimmed milk in PBS-Tween at room temperature for 2 h. Incubated the membranes with diluted primary antibodies at 4 °C for overnight. And the diluted HRP-conjugated secondary antibodies were incubated at room temperature for 1 h. Finally, protein visualization was performed using BeyoECL Star (Beyotime). Immunoblot images were analyzeds with Image Lab software (Bio-Rad Laboratories, Inc.). The antibodies used in this study are listed in Table S3.

5hmC Enzyme-linked immunosorbent assay (ELISA)

Total DNA was extracted using TIANamp Genomic DNA kit (TIANGEN). DNA concentration and purity were measured using a NanoDrop instrument. 5hmC level was detected by MethylFlash Global DNA Hydroxymethylation ELISA Easy Kit (Epigentek). In brief, according to manufacturer instructions, 100 ng sample DNA is adding to strip-wells firstly. After incubated at 37 °C for 1 h and three times washing, prepared 5-hmC Detection Complex Solution was added and incubated at room temperature for 50 min. Next, wells were washed five times. Then, a color developer solution was added. Absorbance at 450 nm was determined.

TET activity assay

For TET activity assay, the input proteins were isolated by EpiQuik Nuclear Extraction Kit (Epigentek) following the manufacture instruction. For TET2 activity assay, to extract TET2 protein, lyse cells in a buffer containing detergents and protease inhibitors, then incubate the lysate with an anti-TET2 antibody to allow binding. Add Protein A/G resin (Thermo Fisher Scientific) to capture the antibody-protein complex, followed by washing to remove non-specific proteins. Elute TET2 from the resin using an elution buffer. The TET enzymes and TET2 enzymes activities were measured using Epigenase 5mC Hydroxylase TET Activity/Inhibition Assay Kit (Colorimetric) (Epigentek). In short, according to the manufacturer’s instructions, the methylated substrate is immobilized onto microplate wells in a stable manner. Active TET enzymes interact with the substrate, catalyzing the conversion of methylated substrate into hydroxymethylated products. These TET-mediated hydroxymethylated products are subsequently detectable using a specific antibody. The proportion or quantity of hydroxymethylated products, indicative of enzyme activity, can be quantitatively assessed through colorimetric analysis by measuring absorbance at a wavelength of 450 nm using a microplate spectrophotometer. For TET2 activity assay, to prepare TET2 enzyme, Flag-tagged TET2-CD proteins were immunoprecipitated using anti-Flag beads and eluted from beads by Flag peptides. Enzyme activity assay was performed using 5mC-Hydroxylase TET Activity/Inhibition Assay Kit.

Lentiviral, plasmid and small interfering RNA (si-RNA) construction, and transfection

The lentiviral vectors carrying shTET2 or empty lentiviral vectors were constructed by Hanheng Biotechnology (Shanghai, China). The lentivirus shRNA against human TET2 was constructed using the lentiviral vector pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO. Lentiviral transduction was performed according to the manufacturer's instructions. The target RNAi sequences are listed in Table S2. Stably transfected cells were selected and maintained for 72 h in the presence of 1.5 μg/mL puromycin (Sigma-Aldrich, MO, USA, P4512). The plasmids of pcDNA3.1-NC and pcDNA3.1-TXNIP were constructed using pcDNA3.1 plasmid vectors by Sangon Co., Ltd. (Shanghai, China). The plasmid vectors were transfected according to the manufacturer instructions. The lentiviral empty vector or empty plasmid served as a negative control. si-TET2 and si-NC were purchased from Hanheng Biotechnology (Shanghai, China). si-RNA transduction was performed according to the manufacturer's instructions. The sequences of si-RNA are listed in Table S2.

Hydroxymethylated DNA immunoprecipitation sequencing (hMeDIP-Seq), RNA-seq and sequencing data analysis

hMeDIP-Seq, RNA-seq and bioinformatics analysis were commissioned to the Cloud-Seq Biotech (Shanghai, China). For hMeDIP-Seq, genomic DNA was isolated using phenol–chloroform, precipitated with ethanol and sonicated to 100–500 bp using Bioruptor (Diagenode). Sonicated DNA was end repaired, A tailed, and ligated to adapters by usingNEBNext® Ultra™ DNA Library Prep Kit (NEB). Then, hMeDIP was performed with a monoclonal antibody against 5-hydroxymethylcytosine by following the standard manufacturer’s protocol (Active Motif). hMeDIP DNA libraries were quantified using Quant-iT PicoGreen dsDNA Kits (Life Technologies) and subjected to high-throughput 150 base paired-end sequencing according to the manufacturers recommended protocol. Raw data were generated after sequencing, image analysis, base calling and quality filtering. After adaptor-trimming and low-quality reads removing by cutadapt (v1.9.2) software, high quality reads were generated. Then the high-quality reads were aligned to reference genome using bowtie2 software (v2.2.4) with default parameters. Peak calling was performed by MACS software (v1.4.2). Differentially hydroxymethylated regions (DhMRs) were identified by diffReps software (1.55.4). The enriched peaks and DhMRs were then annotated with the latest UCSC RefSeq database to connect the peak information with the gene annotation. And the enriched peaks were visualized on UCSC Genome Browser. For RNA-seq, total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA), and subjected to mRNA isolation using GenSeq® mRNA Purification Kit (GenSeq, Inc., Shanghai, China). Then, the purified mRNA was used for library construction with GenSeq® Directional RNA Library Prep Kit (GenSeq, Inc., Shanghai, China) by following manufacturer’s recommendations. The mRNA was fragmented into ~ 300 nt in length. The first-strand cDNA was synthesized from the RNA fragments by reverse transcriptase and random hexamer primers, and the second-strand cDNA was synthesized in 2nd Strand Synthesis Buffer with dUTP Mix. Then, the double stranded cDNA fragments were subjected to end-repair and detailing, followed by adapter ligation. The adapter-ligated DNA samples were PCR amplified and purified to obtain sequencing libraries. Finally, the libraries were sequenced with sequencer on the paired-end 150 bp mode.

Cell cycle analysis

A Cell Cycle and Apoptosis Analysis Kit (Beyotime) was used for cell cycle analysis. According to the manufacturer's instructions, treated cells were washed with PBS. Then, collected cells fixed with cold (4 °C) 70% ethanol for 24 h at 4 °C. After fixation, the cells was washed by PBS. Next, prepared PI/RNase Staining MIX Buffer was added and incubated in 37 °C for 30 min. The DNA content was detected by NovoCyte Advanteon flow cytometer (Agilent). Novoexpression software was used for analysis.

Cell proliferation assay

The Cell Counting Kit-8 (CCK8) (MCE) was used for detected cell proliferation. Following the manufacturer's instructions, shortly, treated cells mixed with 10 μl CCK8 solution in 96 well plate. Culture plate Incubated in the incubator for 2 h. Absorbance at 450 nm was determined.

hMeDIP-qPCR

Total DNA was extracted by TIANamp Genomic DNA kit (TIANGE). DNA concentration and purity were measured using a NanoDrop instrument. 5hmC level of TXNIP was measured by hMeDIP-qPCR. Briefly, the genomic DNA was ultrasound processed to fragments of about 200–800 bp. After breaking, incubate at 99 °C for 10 min to obtain single-stranded DNA. And retain 10% of the total volume of genomic DNA fragments as input. The remaining DNA fragments were immunoprecipitated with anti-5hmC antibody at 4 °C for 3 h. After incubation, the DNA antibody complex was captured with G protein magnetic beads at 4 °C for 3 h. The immunoprecipitated DNA was purified using the MinElute PCR purification kit (Qiagen). The enriched DNA was amplified by qPCR, and the corresponding 5hmC enrichment was normalized using input. The primers used in hMeDIP-qPCR are listed in Table S2.

Chromatin immunoprecipitation qPCR (CHIP-qPCR)

A EZ ChIP™ Chromatin Immunoprecipitation Kit (Sigma-Aldrich) was used for CHIP-qPCR. Shortly, according to the manufacturer's instructions, collected cells are treated in formaldehyde for 10 min. The cell lysates were then ultrasonically treated to obtain chromatin fragments of 200-300 bp and immunoprecipitated with anti-TET2 antibody and control IgG. qPCR was performed on the recovered precipitated chromatin DNA. The primers used in CHIP-qPCR are listed in Table S2.

Statistical analysis

Statistical analyses and data visualization were performed using GraphPad Prism version 9.4 (GraphPad Software, Inc.) or R software version 4.0.3. The data are presented as mean ± SD/SEM or median. For the comparison between the two groups, Student’s t-test or the Wilcoxon rank-sum test was used, depending on whether the data followed a normal distribution. The comparisons between more than two groups were analyzed using one-way analysis of variance (ANOVA) or the Kruskal–Wallis test. The correlation analysis was conducted using Pearson correlation. Survival curves were generated using the Kaplan–Meier method and significance assessed using the log rank test. All statistical tests were two-tailed, and p-value < 0.05 was considered as significant (*p < 0.05, **p < 0.01, ***p < 0.001).

The GSE14879 are sourced from the GEO database, with the downloaded data in MINiML format, which includes all platforms, samples, and complete GSE records in the GSE. We use the normalize quantiles function in the preprocessCore package of R for data standardization and the remove BatchEffect function in the limma package of R to remove batch effects. For data preprocessing results, we evaluate the data normalization situation through box plots; the situation of data batch effects is evaluated by comparing the PCA plots before and after batch removal.

Results

Decreased TET2 induced lower 5hmC modification level in in anaplastic large cell lymphoma patients

To investigate the role of 5hmC modification in the lymphomagenesis of ALCL, IHC staining for 5hmC modification was conducted on a total of 46 FFPE samples from ALCL patients. Additionally, 24 samples of reactive lymph nodes were utilized as a control group. IHC staining revealed a reduction in the levels of 5hmC in patients with ALCL compared to the control group (Fig. 1A, B). However, the 5hmC levels were not significantly different between ALK + ALCL samples and ALK- ALCL samples (Fig S1A). To identify the protein responsible for reduced 5hmC levels, we downloaded the GSE14879 dataset from the Gene Expression Omnibus (GEO) database. We evaluated the expression levels of TET1, TET2, TET3, IDH1, IDH2, IDH3A, IDH3B. The results indicated reduced expression levels of TET2 and IDH2 in ALCL samples compared to CD30 + T cells (Fig S1B). To confirm the result, IHC staining for TET2 and IDH2 was conducted, which revealed a decrease in the expression levels of both proteins (Fig. 1C, 1D, S1D, S1E). The expression levels of these were not significantly different between the ALK + group and the ALK- ALCL group, either (Fig S1C, S1E). To further screen the protein associated with 5hmC modification in ALCL, we analyzed the correlation between their respective IHC scores and the 5hmC IHC score. The outcomes revealed that TET2 expression was related to 5hmC modification in ALCL (Fig. 1E, 1F, Fig S1F, G). The overall survival of 5hmC-negative group was lower than in 5hmC-positive group (Fig. 1G). Similarly, TET2-negative group had lower overall survival compared to TET2-positive group (Fig. 1H). In general, in the context of ALCL, low-level 5hmC modification correlates with TET2 expression.

Fig. 1
figure 1

Decreased TET2 mediates low level of 5hmC in anaplastic large cell lymphoma patients. A Representative IHC staining of 5hmC in ALCL samples(n = 46) and reactive lymph nodes (n = 24). B IHC score analysis for 5hmC in in ALCL samples(n = 46) and reactive lymph nodes (n = 24). C Representative IHC staining of TET2 in ALCL samples (n = 46) and reactive lymph nodes (n = 24). D IHC score analysis for TET2 in ALCL samples (n = 46) and reactive lymph nodes (n = 24). E Representative IHC staining of 5hmC and TET2 with different levels in ALCL samples (n = 46). F Correlation of 5hmC expression and TET2 expression in ALCL samples (n = 46). G Kaplan–Meier survival curve of ALCL patients with different levels of 5hmC IHC score (n = 46). H Kaplan–Meier survival curve of ALCL patients with different levels of TET2 IHC score (n = 46). 5hmC, 5-Hydroxymethylcytosine, IHC, immunohistochemistry; ALCL, anaplastic large cell lymphoma. Data were expressed as the mean ± SDs. Representative images were captured at × 400 magnification. *p < 0.05; *p < 0.01;***p < 0.001

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Knockdown of TET2 promoted the cell cycle progress and proliferation

ALCL patients with varying ALK statuses demonstrates substantial differences in pathogenesis, therapeutic approaches, and prognosis. To further explore the role of TET2 in ALCL, we used Su-DHL-1 (ALK +) and DL-40 (ALK-) cell lines as in vitro models, utilizing normal T cells isolated from healthy donors as controls. Firstly, we measured the 5hmC modification level in Su-DHL-1 and DL-40. The analysis demonstrated a reduction 5hmC level in the ALCL cell lines compared to normal T cells (Fig. 2A). Similarly, there was a concomitant decrease in TET2 expression (Fig. 2B, 2C). For the purpose of further investigating the function of TET2 in ALCL, stable knockdown TET2 ALCL cell lines were constructed. The knockdown efficiency was assessed by real-time qPCR and Western blot (Fig. 2D, 2E). The 5hmC modification was decreased after TET2 knockdown (TET2 KD) (Fig. 2F). Then, we conducted RNA-seq on TET2 KD ALCL cell lines. KEGG pathway analysis were used to enrich and analyze differentially expressed gene sets. The results indicated the cell cycle pathway was the most related to TET2 KD (Fig. 2G). Gene set enrichment analysis (GSEA) indicates that genes involved in the G1/S transition of the mitotic cell cycle are significantly enriched at the upper end of the ranked gene list, suggesting that this pathway is potentially activated or upregulated. (Fig. 2H). The volcano plots illustrated these transcripts, with the red/blue points (upregulated/downregulated) representing statistically significant differences in gene expression. These differences were determined using a false discovery rate (FDR) < 0.05 and fold change > 2 as the cutoff value (Fig S2A). To verify these conclusions, the cell cycle was measured by flow cytometry. The experiment results revealed a reduction in cells in the G1 phase and accumulation of cells in the S phase compared to normal control in TET2 ALCL cell lines (Fig. 2I). Similarly, the ability of cell proliferation was increased after TET2 knockdown (Fig. 2J). To further validate our findings, we conducted siRNA knockdown experiments. The results consistently demonstrated that reduced TET2 expression promotes cell cycle progression and proliferation (Fig S2B-2F). Overall, TET2 knockdown was shown to enhance both cell proliferation and cell cycle progression.

Fig. 2
figure 2

TET2 knockdown promotes cell cycle and proliferation. A The 5hmC levels in normal T cells, Su-DHL-1 and DL-40 were determined by ELISA. B The TET2 mRNA expression in normal T cells, Su-DHL-1and DL-40 was determined by qPCR. C The TET2 protein expression in normal T cells, Su-DHL-1 and DL-40 was determined by Western blot. D Validation of TET2 knockdown in ALCL cell lines was determined by qPCR. E Validation of TET2 knockdown in ALCL cell lines was determined by Western blot. F The 5hmC levels in ALCL cell lines with TET2 knockdown or negative control were determined by ELISA. G KEGG pathway analysis of DEGs in ALCL cell lines with TET2 knockdown or negative control. H GSEA analysis of G1/S transition of mitotic cell cycle. I The cell cycle distribution of ALCL cell lines with TET2 knockdown or negative control was determined by flow cytometry. J The cell proliferation ability of ALCL cell lines with TET2 knockdown or negative control was determined by the CCK-8 assay. ELISA, Enzyme linked immunosorbent assay, KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, Gene set enrichment analysis; DEGs, Differential Gene Expression, CCK8, Cell Counting Kit-8. All experiments were repeated three times independently (n = 3). Data were expressed as the mean ± SDs. *p < 0.05; **p < 0.01; ***p < 0.001

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TET2-modified 5hmC influenced cell cycle progression and proliferation

L-Ascorbic acid (L-AA) promoted the modification of 5hmC. To probe the effect of 5hmC in cell cycle progression in ALCL cell lines, the cells were treated with L-AA. Valuation of 5hmC level was performed by ELISA after L-AA treatment. As previously reported [15], L-AA treated groups had increased 5hmC level compared to PBS-treated control group (Fig. 3A). While the activity of TET and TET2 enzymes was increased (Fig S3A, Fig. 3B). Also, treatment with L-AA promoted cell cycle arrest of ALCL cells. The percentage of G1 phase was increased while the S phase was decreased (Fig. 3C). Furthermore, the cell proliferation ability of ALCL cell lines was suppressed by L-AA (Fig. 3D). Besides, the expression of TET2 in L-AA treated group was not significant difference with control group (Fig S3B, S3C). Bobcat339 is a recognized inhibitor to TET enzymes. After treatment with Bobcat339, the activity of TET and TET2 enzymes was significantly decreased (Fig S3D, Fig. 3E). Meanwhile, the 5hmC level of Bobcat339-treated group was reduced compared to DMSO-treated control group (Fig. 3F). Flow cytometry was used for detecting cell cycle progression of these groups. As expected, Bobcat339 promoted the cell cycle of ALCL cell lines by increasing the proportion of cells in the S phase but decreasing cells in the G1 phase (Fig. 3G). Also, cell proliferation was enhanced following Bobcat399 treatment. (Fig. 3H). Additionally, TET2 expression analysis did not show any significant differences between the control group and the experimental group (Fig S3E, S3F). To strengthening the correlation between 5hmC changes and TET2 in ALCL, we using L-AA on TET2 KD and NC. The results shown, compared with NC, TET2 KD with L-AA treatment resulted in a lower increase in 5hmC levels (Fig S3G).

Fig. 3
figure 3

Effects of L-Ascorbic acid and Bobcat339 on ALCL cell lines. A The 5hmC levels of ALCL cell lines after L-AA treatment were determined by ELISA, compared with PBS-treated control. B TET2 enzymatic activity of ALCL cell lines after L-AA treatment was determined by ELISA, compared with PBS-treated controls. C The cell cycle distribution of ALCL cell lines after L-AA treatment was determined by flow cytometry, compared with PBS-treated controls. D The cell proliferation ability of ALCL cell lines after L-AA treatment was determined by the CCK-8 assay, compared with PBS-treated controls. E TET2 enzymatic activity of ALCL cell lines after Bobcat339 treatment was determined by ELISA, compared with DMSO-treated controls. F The 5hmC levels of ALCL cell lines after Bobcat339 treatment were determined by ELISA, compared with DMSO-treated controls. G The cell cycle distribution of ALCL cell lines after Bobcat339 treatment was determined by flow cytometry, compared with DMSO-treated controls. H The cell proliferation ability of ALCL cell lines after Bobcat339 treatment was determined by the CCK-8 assay, compared with DMSO-treated controls. L-AA, L-Ascorbic acid; DMSO, Dimethyl sulfoxide. Data were expressed as the mean ± SDs. All experiments were repeated three times independently (n = 3). *p < 0.05;**p < 0.01;***p < 0.001

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TXNIP was the downstream target molecule of TET2 in ALCL cell lines.

hMeDIP-seq was conducted to search for the putative downstream component of TET2. Based on the average of all the peaks obtained by hMeDIP-seq, we found that 5-hmC peaks in TET Knockdown ALCL cells were less intense than those in control ALCL cells (Fig. 4A). After integrating hMeDIP-seq results, RNA-seq results, and the set of cell cycle-related genes (GO: 0051301, GO: 0007049), 23 transcripts overlapped (Fig. 4B). Genes with a |log2 Fold Change|> 2 were selected for further verification (Fig S4A-S4C). Results of analysis proved that expression of TXNIP was remarkably decreased upon TET2 knockdown (Fig. 4C, D). According to hMeDIP-seq data, there were differences in peak intensities in TXNIP gene from chr1: 145,994,001 to 145,994,380 in ALCL cells with TET2 knockdown compared to those without (Fig. 4E). With TET2 knockdown, there was lower 5hmC enrichment and reduced TET2 recruitment on the TXNIP gene (Fig. 4F, G). To further explore the connection between 5hmC modification and TXNIP, we assessed TXNIP expression following L-AA and Bobcat339 treatments. The results revealed that L-AA treatment elevated TXNIP expression levels, whereas Bobcat339 treatment led to a reduction in TXNIP expression (Fig S4D, S4E). Next, the expression level of TXNIP was determined by IHC staining in ALCL samples and reactive lymph nodes. Analysis revealed a reduction in expression TXNIP level in ALCL samples compared to controls (Fig. 4H, I). Furthermore, this reduction was associated with TET2 expression (Fig. 4J). Overall survival of TXNIP-negatives showed no statistical difference from TXNIP-positives (p > 0.05) (Fig S4F).

Fig. 4
figure 4

Decreased TET2 expression led to a reduction in TXNIP levels by downregulating 5hmC levels. A 5hmC coverage depth (per base pair per peak per 10 million mapped reads) of 5hmC peaks (− 2 kb to + 2 kb) in TET2-knockdown ALCL cell lines and controls. B The Venn diagram of genes associated with downregulated enrichment peaks in RNA-seq from TET2 knockdown ALCL cell lines, 5hmC downregulated genes identified through hMeDIP-seq in TET2 knockdown ALCL cell lines, and cell cycle-related gene sets (GO: 0051301, GO: 0007049). C The TXNIP mRNA expression in TET2 knockdown ALCL cell lines and controls was determined by qPCR (n = 3). D The TXNIP protein expression in TET2 knockdown ALCL cell lines and controls was determined by Western blot (n = 3). E Representative 5hmC sites in TXNIP genes represented by integrative genomics viewer. F 5hmC enrichment in TXNIP of TET2 knockdown ALCL cell lines and controls measured by hMeDIP-qPCR (n = 3). G The Analysis of TET2 recruitment at the TXNIP gene in TET2 knockdown ALCL cell lines and controls was determined by CHIP-qPCR (n = 3). H Representative IHC staining of TXNIP with in ALCL samples (n = 46) and reactive lymph nodes (n = 24). I IHC score analysis for TXNIP in ALCL samples (n = 46) and reactive lymph nodes (n = 24). J Correlation of TXNIP expression and TET2 expression in ALCL samples (n = 46). hMeDIP, Hydroxymethylated DNA Immunoprecipitation; CHIP, Chromatin Immunoprecipitation. Data were expressed as the mean ± SDs.  *p < 0.05; **p < 0.01; ***p < 0.001

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TXNIP involved in cell cycle progression and proliferation in ALCL cell lines

To investigate the mechanism through which TXNIP regulates the cell cycle in ALCL cell lines, alterations in cell cycle-related factors were analyzed. The results indicated that CDK2, CDK4, and CDK6 were upregulated. Furthermore, there was an increase in the phosphorylation of Rb, whereas the phosphorylation of p53 was decreased (Fig. 5A). Besides, the expression of p21 was decreased, either (Fig. 5B). Subsequently, we transfected the TET2 knockdown ALCL cell lines with the pcDNA3.1-TXNIP plasmid and the pcDNA3.1 control plasmid (pcDNA3.1-NC). Following treatment with pcDNA3.1-TXNIP, there was a notable upregulation of TXNIP expression in the pcDNA3.1-TXNIP group, whereas the expression levels of TET2 remained unchanged (Fig S5A, B). Also, the TET2 activity showed no variation (Fig S5C). Next, we analyzed the cell cycle structure of these groups. The experiment demonstrated that the overexpression of TXNIP impeded the progression of the cell cycle from the G1 phase to the S phase (Fig. 5C). Consequently, cell proliferation was also inhibited (Fig. 5D). Alterations were observed in expression of cell cycle-related factors after overexpression TXNIP. Specifically, the levels of CDK2, CDK4, CDK6 and phosphorylated Rb were reduced, whereas the phosphorylated p53 was enhanced, as demonstrated in the results (Fig. 5E). The expression of p21 was increased, either (Fig. 5F). To further investigate the role of p53 in TXNIP-mediated regulation of the cell cycle, we employed Pifithrin-α (PFTα), a p53 inhibitor, to validate our findings. Following transfection with DNA3.1-TXNIP, cells were treated with PFTα, which resulted in decreased p53 levels while TXNIP expression remained unchanged (Fig S5D). The results demonstrated that while PFTα treatment promoted cell cycle progression and enhanced cell proliferation (Fig. 5G, 5H). The expression of CDK2, CDK4, and CDK6 were upregulated. Besides, there was an increase in the phosphorylation of Rb, whereas the phosphorylation of p53 was decreased (Fig S5E). The expression of p21 was decreased after PFTα treatment. (Fig S5F). The changes in the expression of cell cycle-related proteins were consistent with our previous findings.

Fig. 5
figure 5

TXNIP blocks cell cycle progression through p53. A The cell cycle-related proteins expression levels in TET2 knockdown ALCL cell lines and controls were determined by Western blot. B The p21 protein expression levels in TET2 knockdown ALCL cell lines and controls were determined by Western blot. C The cell cycle distribution of ALCL cell lines transfected with pcDNA3.1-TXNIP or pcDNA3.1-NC was determined by flow cytometry. D The cell proliferation ability of ALCL cell lines transfected with pcDNA3.1-TXNIP or pcDNA3.1-NC was determined by the CCK-8 assay. E The cell cycle-related proteins expression levels in TET2 knockdown ALCL cell lines transfected with pcDNA3.1-TXNIP or pcDNA3.1-NC were determined by Western blot. F The p21 protein expression levels in TET2 knockdown ALCL cell lines transfected with pcDNA3.1-TXNIP or pcDNA3.1-NC were determined by Western blot. G The cell cycle distribution of ALCL cell lines treated with PFTα or DMSO control after transfection with pcDNA3.1-TXNIP was determined by flow cytometry. H The cell proliferation ability of ALCL cell lines treated with PFTα or DMSO control after transfection with pcDNA3.1-TXNIP was determined by the CCK-8 assay. P, phosphorylation. All experiments were repeated three times independently (n = 3). Data were expressed as the mean ± SDs. * p < 0.05;  ** p < 0.01;  *** p < 0.001

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Discussion

In this study, we identified TET2 as a key contributor to the reduced 5hmC levels observed in ALCL. Furthermore, the 5hmC modification of the TXNIP gene, induced by a reduction in TET2 levels, influenced TXNIP expression, which in turn promoted cell cycle progression and proliferation in ALCL cell lines. These findings hold promise for the development of novel therapeutic strategies for ALCL.

The role of 5hmC in regulating gene expression is becoming clearer. 5hmC is not merely a passive marker of DNA methylation but an active participant in gene expression regulation, particularly in the context of cancer [16]. Recent studies have demonstrated that the loss of 5hmC is a common epigenetic hallmark of cancer. For instance, in melanoma, genome-wide mapping revealed a significant reduction in the 5hmC landscape, correlating with poor prognosis and tumor progression [17]. Similarly, in glioblastoma, the downregulation of TET3 has been associated with reduced 5hmC levels, promoting tumorigenesis. The epigenetic repression of TET3 has also been shown to correlate with worse outcomes in glioma patients, suggesting that 5hmC levels could serve as a prognostic marker [18]. Overall, growing evidence indicates that 5hmC modification plays a crucial role in various cancer pathologies. Therefore, further studies are essential to determine whether 5hmC modification also plays a causative role in ALCL.

In this study, we initially quantified the levels of 5hmC in ALCL samples and reactive lymphoid nodes. IHC staining results revealed reduced levels of 5hmC in ALCL samples, consistent with previous studies [14]. Next, we evaluated the expression of 5hmC-related enzymes in ALCL samples and CD30 + T cells derived from the GSE14879 dataset. Analysis revealed decreased levels of TET2 and IDH2, which we confirmed through IHC staining. However, IHC scores showed that only TET2 expression levels positively correlated with 5hmC levels. Additionally, the IHC scores of TET2 and 5hmC could serve as prognostic factors in ALCL patient prognosis, alongside known clinical prognostic factors [19,20,21]. Current reports suggest that reductions in 5hmC levels are not solely attributable to decreased 5mC levels. Instead, multiple pathways may contribute to the observed reduction in 5hmC levels [22, 23]. Therefore, our study focused specifically on 5hmC modification, without assessing 5mC levels. The relationship between 5mC and 5hmC in ALCL will be investigated in future studies.

ALCL patients with varying ALK statuses demonstrate substantial differences in pathogenesis, therapeutic approaches, and prognosis. To validate our conclusions, we used Su-DHL-1 and DL-40 as in vitro models. The findings of decreased 5hmC and TET2 levels in clinical samples were consistent with the results observed in these cell lines. To further investigate the relationship between TET2 and 5hmC modification, we constructed stable TET2-knockdown ALCL cell lines. A lower level of 5hmC was observed in the knockdown cells. RNA sequencing results revealed a strong correlation between cell cycle progression and reduced TET2 levels. This conclusion aligns with previous studies and is further supported by our flow cytometry analysis of cell cycle distribution [13]. Cell cycle regulation is a critical process that governs cell proliferation [24, 25]. Hence, we measured the cell proliferation ability of the control and experimental groups. The experimental results indicated that cell proliferation was enhanced after TET2 knockdown, as expected. In short, reduced TET2 levels led to decreased 5hmC modification in ALCL cells, promoting cell cycle progression and proliferation.

TET2 is a member of the TET family of dioxygenases, which plays a crucial role in the regulation of DNA methylation and demethylation processes. TET2 specifically catalyzes the conversion of 5mC to 5hmC, a key step in the active demethylation pathway that influences gene expression. TET2 is increasingly recognized as a significant player in the pathogenesis of various lymphomas, particularly due to its role in DNA methylation and epigenetic regulation. Mutations in TET2 are frequently observed in hematologic malignancies, including diffuse large B cell lymphoma (DLBCL) and T cell lymphomas. These mutations can lead to impaired 5hmC, which is crucial for normal myelopoiesis and B cell differentiation [26, 27]. Moreover, TET2 mutations are also implicated in T cell malignancies, such as angioimmunoblastic T cell lymphoma (AITL) and peripheral T cell lymphoma. In these contexts, TET2 loss-of-function mutations contribute to the overproduction of follicular helper T cell-like populations, which can lead to lymphomagenesis [28]. While our earlier study found TET2 inactivation mutations in 3 out of 21 ALK- ALCL cases [29]. The presence of TET2 mutations in both myeloid and lymphoid malignancies suggest a shared pathogenic mechanism that may arise from early progenitor cells [30]. In summary, TET2 plays a critical role in the development and progression of various lymphomas through its influence on DNA methylation and gene expression [31, 32].

In the context of hematological malignancies, TET2 has been shown to influence cell cycle dynamics. For instance, studies have demonstrated that TET2 overexpression in chronic lymphocytic leukemia (CLL) blocks cell cycle progression [33]. Moreover, the knockdown of TET2 in ENKTL cell lines has been linked to prolonged DNA synthesis periods and increased cloning ability [13]. The balance between 5mC and 5hmC levels, modulated by TET2, appears to correlate with various clinical parameters, including disease stage and patient survival, further underscoring its significance in cell cycle regulation. Additionally, the broader implications of TET2 in cell cycle regulation can be observed in its interactions with other cell cycle regulators. For example, TET2 may influence the expression of genes involved in the cell cycle, thereby affecting the activity of cyclin-dependent kinases (CDKs) and their regulatory partners [34]. In general, TET2 plays a critical role in the regulation of the cell cycle through its epigenetic functions and interactions with other cell cycle regulators.

To examine the relationship among 5hmC, TET2, and cell cycle progression in depth, we treated cells with L-AA or Bobcat339. L-AA, commonly known as vitamin C, is involved in the regulation of the 5hmC modification [15, 35]. The experimental results suggested that an increase in 5hmC modification promoted cell cycle arrest and inhibited cellular proliferation, without altering TET2 expression level but TET2 enzymes activity. Bobcat339 was recognized as a TET enzyme inhibitor [36]. Our research found that Bobcat339 treatment raised S phase cells and lowered G1 phase cells by reducing 5hmC levels and TET2 activity. In conclusion, TET2-mediated 5hmC modification affects cell cycle progression and proliferation.

The mechanism of TET2 affecting cell cycle progression through 5hmC modification has not been reported. For further investigation, we conducted hMeDIP-seq. By integrating sequencing data with cell cycle-related GO annotations and RNA-seq results, we identified TXNIP as the target gene whose expression was altered by decreased 5hmC modification following TET2 knockdown. The regulation of TXNIP by TET2 has been previously identified in studies on diabetes. The studies demonstrated that reduced TET2 expression in diabetic mouse models led to a significant decrease in TXNIP levels [37]. This finding aligns with our conclusion that a reduction in TET2 results in decreased TXNIP expression.

Thioredoxin-interacting protein (TXNIP) plays a crucial role in various biological processes, particularly in the regulation of oxidative stress and inflammation [38]. It acts as a negative regulator of thioredoxin, an important antioxidant, thereby influencing cellular redox status. TXNIP has emerged as a significant player in the pathophysiology of various lymphomas. In mantle cell lymphoma (MCL), TXNIP has been identified as a potential tumor suppressor. Studies show that TXNIP expression was downregulated in MCL, and this downregulation correlates with aggressive disease features and poor prognosis [39]. Moreover, in T cell lymphomas, its expression is influenced by inflammatory cytokines, which can alter T cell activation and survival [40]. Overall, TXNIP serves as a critical regulator in the pathogenesis of lymphomas, influencing immune response and treatment resistance.

Finally, our studies discussed the mechanism of TXNIP that block the cell cycle progression. As the result shown, expression of TXNIP altered the level of P-p53 which caused the changes of cell cycle related proteins. Additionally, the decreased activity of p53 induces cell cycle arrest at the G1/S phase through the p53-p21-CDK2-Rb pathway [41]. The downregulation of the cell cycle by TXNIP has been reported. The expression level of TXNIP is often decreased in tumor cells, leading to changes in cell cycle dynamics. Recent studies have shown that TXNIP can influence the cell cycle by regulating key CDK inhibitors p21 [42]. Moreover, it has been shown to mediate differential responses to various treatments in cancer cells, such as sodium butyrate and sodium 4-phenylbutyrate, where TXNIP expression levels dictate the extent of cell death and cell cycle arrest [43]. Furthermore, the expression of TXNIP is usually downregulated in breast cancer, where its loss is associated with high proliferative activity and estrogen-dependent growth, suggesting that TXNIP functions as a tumor suppressor by inhibiting excessive cell cycle progression [44]. In summary, our results demonstrated a relationship between TXNIP and cell cycle, consist with previous reports.

Conclusions

To sum up, the current study offers experimental evidence indicating that 5hmC modification, facilitated by TET2, can enhance cell cycle progression in ALCL by modulating TXNIP 5hmC modification (Fig. 6). This finding suggests that 5hmC plays a role in the pathogenesis of ALCL, and targeting TET2 or TXNIP may represent a potential therapeutic strategy for the treatment of ALCL.

Fig. 6
figure 6

Schematic diagram. The schematic of TET2-mediated 5hmC modification of TXNIP promotes the ALCL cell cycle progression and proliferation, as created by Figdraw

Full size image

Availability of data and material

The raw sequence data of hMeDIP-seq and RNA-seq reported in this paper have been deposited in the Genome Sequence Archive (GSA) in National Genomics Data Center, China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA009190) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human. All relevant data are available upon reasonable request.

Abbreviations

5hmC:

5-Hydroxymethylcytosine

ALCLq:

Systemic anaplastic large cell lymphoma

FFPE:

Formalin-fixed, paraffin-embedded

TET:

Ten-eleven translocation

hMeDIP:

Hydroxymethylated DNA Immunoprecipitation

TXNIP:

Thioredoxin interacting protein

5mC:

5-Methylcytosine

2-HG:

2-Hydroxyglutarate

IDH:

Isocitrate dehydrogenase

α-KG:

α-Ketoglutarate

ENKTL:

Extranodal NK/T-cell lymphoma

IHC:

Immunohistochemical

L-AA:

L-Ascorbic acid

CCK8:

Cell Counting Kit-8

CHIP-qPCR:

Chromatin immunoprecipitation qPCR

GEO:

Gene Expression Omnibus

KEGG:

Kyoto Encyclopedia of Genes and Genomes

DMSO:

Dimethyl sulfoxide

DLBCL:

Diffuse large B-cell lymphoma

AITL:

Angioimmunoblastic T-cell lymphoma

CLL:

Chronic lymphocytic leukemia

CDKs:

Cyclin-dependent kinases

MCL:

Mantle cell lymphoma

ROS:

Reactive oxygen species

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Acknowledgements

We sincerely thank all the patients for their participation.

Funding

This work was supported by Joint Funds for the innovation of science and Technology Fujian province (Grant number: 2021Y9215) and Non-research Project Funds of Fujian Provincial Cancer Hospital (Grant number: F2326Y-YZK06-01).

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Author notes

  1. #Ziqing Yu and Lihua Zhong These authors contributed equally to this work and should be considered co-first authors.

Authors and Affiliations

  1. Department of Pathology, Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital, Fuzhou, China

    Ziqing Yu, Lihua Zhong, Wangyang Tang, Wenfang Zhang, Ting Lin, Weifeng Zhu, Gang Chen & Jianchao Wang

  2. Department of Pathology, Sir Run Run Shaw Hospital (SRRSH), affiliated with the Zhejiang University School of Medicine, Hangzhou, China

    Wangyang Tang

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Contributions

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. J.W and G.C have complete access to the study data and are responsible for its integrity and analysis accuracy. Z.Y and L.Z developed the concept, designed and conducted most experiments, and wrote the manuscript. W.T contributed to the experiments and manuscript preparation, while T.L, W.Zhu and W.Zhang assisted with collecting clinical samples. All authors reviewed the manuscript.

Corresponding authors

Correspondence to Gang Chen or Jianchao Wang.

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The present study was approved by Institutional Medical Ethics Review Board of Fujian Cancer Hospital. The authors declare no violation of the Helsinki Doctrine on human experimentation. Verbal and written informed consent were obtained from all participants.

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Supplementary Information

13148_2025_1816_MOESM1_ESM.tif

Additional file1 (Figure S1: Low expression of TET2 induces decreased level of 5hmC in anaplastic large cell lymphoma patients. (A) IHC score analysis for 5hmC in ALK+ ALCL samples (n=7) and ALK- ALCL samples (n=39). (B) The relative expression level of 5hmC-modified genes in ALCL samples (n=9) and CD30+ cells (n=5) based on GSE14879. (C) IHC score analysis for TET2 in ALK+ ALCL samples (n=7) and ALK- ALCL samples (n=39). (D) Representative IHC staining of IDH2 in ALK+ ALCL samples (n=7), ALK- ALCL samples (n=39) and reactive lymph nodes (n=24). (E) IHC score analysis for IDH2 in ALK+ ALCL samples (n=7), ALK- ALCL samples (n=39), and reactive lymph nodes (n=24). (F) Representative IHC staining of 5hmC and IDH2 with different levels in ALCL samples (n=46). (G) Correlation of 5hmC expression and TET2 expression in ALCL samples (n=46). Data were expressed as the mean ± SDs. Representative images were captured at ×400 magnification. *p < 0.05; **p < 0.01; ***p < 0.001.)

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Additional file2 (Figure S2: TET2 knockdown with si-RNA promotes cell cycle progression and proliferative ability in ALCL cell lines. (A) Volcano map of differential expression mRNAs in ALCL cell lines with TET2 knockdown or negative controls. (B) The TET2 mRNA expression level of ALCL cell lines transfected with si-TET2 or si-NC were determined by qPCR (n=3). (C) The 5hmC levels in ALCL cell lines transfected with si-TET2 or si-NC were determined by ELISA (n=3). (D) The TET2 protein expression level of ALCL cell lines transfected with si-TET2 or si-NC were determined by Western blot (n=3). (E) The cell cycle distribution of ALCL cell lines transfected with si-TET2 or si-NC was determined by flow cytometry. (n=3) (F) The cell proliferation ability of ALCL cell lines transfected with si-TET2 or si-NC was determined by the CCK-8 assay (n=3). Data were expressed as the mean ± SDs. *p < 0.05; **p < 0.01; ***p < 0.001.)

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Additional file3 (Figure S3: Effects of L-Ascorbic acid and Bobcat339 on TET2 of ALCL cell lines. (A) TET enzymatic activity of ALCL cell lines after L-AA treatment was determined by ELISA, compared with PBS-treated controls. (B) The TET2 mRNA expression in ALCL cell lines after L-AA treatment was determined by qPCR, compared with PBS-treated control. (C) The TET2 protein expression in ALCL cell lines after L-AA treatment was determined by Western blot, compared with PBS-treated control. (D) TET enzymatic activity of ALCL cell lines after Bobcat339 treatment was determined by ELISA, compared with DMSO-treated controls. (E) The TET2 mRNA expression in ALCL cell lines after Bobcat339 treatment was determined by qPCR, compared with DMSO-treated control. (F) The TET2 protein expression in ALCL cell lines after Bobcat339 treatment was determined by Western blot, compared with DMSO-treated control. (G) The 5hmC levels in ALCL cell lines with TET2 knockdown and control after L-AA treatment were determined by ELISA, compared with PBS-treated controls. All experiments were repeated three times independently (n=3). Data were expressed as the mean ± SDs. *p < 0.05; **p < 0.01; ***p < 0.001.)

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Additional file4 (Figure S4: TXNIP is a downstream target of TET2. (A) The relative mRNA expression of candidate genes in Su-DHL-1 cell line determined by qPCR (n=3). (B) The relative mRNA expression of candidate genes in DL-40 cell line determined by qPCR (n=3). (C) The protein expression levels of candidate genes ALCL cell lines determined by Western blot (n=3). (D) The TXNIP protein expression levels of ALCL cell lines after L-AA treatment were determined by Western blot, compared with PBS-treated controls. (E) The TXNIP protein expression levels of ALCL cell lines after Bobcat339 treatment were determined by Western blot, compared with DMSO-treated controls. (F) Kaplan-Meier survival curve of ALCL patients with different levels of TXNIP IHC score (n=46). Data were expressed as the mean ± SDs. *p < 0.05; **p < 0.01; ***p < 0.001.)

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Additional file5 (Figure S5: TXNIP promoted the cell cycle arrest. (A) The TXNIP mRNA expression levels of ALCL cell lines transfected with pcDNA3.1-TXNIP or pcDNA3.1-NC were determined by qPCR. (B) The TXNIP protein expression levels of ALCL cell lines transfected with pcDNA3.1-TXNIP or pcDNA3.1-NC were determined by Western blot. (C) TET2 enzymatic activity of TET2 knockdown ALCL cell lines transfected with pcDNA3.1-TXNIP or pcDNA3.1-NC was determined by ELISA, compared with PBS-treated controls. (D) The TXNIP and p53 protein expression levels of ALCL cell lines treated with PFTα or DMSO control after transfection with pcDNA3.1-TXNIP were determined by Western blot. (E) The cell cycle-related proteins expression levels in ALCL cell lines treated with PFTα or DMSO control after transfection with pcDNA3.1-TXNIP were determined by Western blot. (F) The p21 proteins expression levels in ALCL cell lines treated with PFTα or DMSO control after transfection with pcDNA3.1-TXNIP were determined by Western blot. All experiments were repeated three times independently (n=3). Data were expressed as the mean ± SDs. *p < 0.05; **p < 0.01; ***p < 0.001.)

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Yu, Z., Zhong, L., Tang, W. et al. TET2-mediated 5-hydroxymethylcytosine of TXNIP promotes cell cycle arrest in systemic anaplastic large cell lymphoma. Clin Epigenet 17, 10 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13148-025-01816-0

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13148-025-01816-0

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Clinical Epigenetics

ISSN: 1868-7083

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