Supplementary Material for: The Thioredoxin-Interacting Protein TXNIP Is a Putative Tumour Suppressor in Cutaneous T-Cell Lymphoma

Background: The thioredoxin-interacting protein (TXNIP) is involved in cellular metabolism and cell proliferation, and recently, deficient expression of TXNIP has been associated with progression and poor outcome for cancer patients. Objectives: To assess TXNIP expression and function in malignant T cells from cutaneous T-cell lymphoma (CTCL). Methods: CTCL-derived malignant (MyLa2059, PB2B) and non-malignant (MyLa1850) cell lines were analysed by Western blotting and qPCR for TXNIP expression. Subsequently, the malignant CTCL cell lines were treated with GSK126 – an inhibitor of enhancer of zeste homolog 2 (EZH2) methyltransferase activity or assessed by bisulphite sequencing for TXNIP promoter methylation. Methylation was also assessed with the demethylating agent 5-azacytidine (5AZA). Finally, TXNIP was overexpressed in the malignant PB2B cell line via plasmid transduction, and the effect of TXNIP was further analysed by flow cytometry. Results: We report on low expression of TXNIP protein in all cell lines representing different subtypes and stages of CTCL when compared to non-malignant T cells. Epigenetic silencing and other mechanisms were involved in the repression of TXNIP whereas forced expression of TXNIP strongly inhibited proliferation of malignant T cells. Conclusions: Epigenetic silencing and other as yet unknown mechanisms repress TXNIP expression in malignant T cells. As forced expression of TXNIP inhibits malignant proliferation, we propose that TXNIP is a putative tumour suppressor in CTCL.背景：硫氧还蛋白相互作用蛋白（thioredoxin-interacting protein, TXNIP）参与细胞代谢与细胞增殖过程，近期研究表明其表达缺失与癌症患者的疾病进展及不良预后密切相关。 研究目的：评估皮肤T细胞淋巴瘤（cutaneous T-cell lymphoma, CTCL）恶性T细胞中TXNIP的表达水平与生物学功能。 实验方法：通过蛋白质印迹法（Western blotting）与实时荧光定量PCR（qPCR），分析源自CTCL的恶性细胞系（MyLa2059、PB2B）及非恶性细胞系（MyLa1850）的TXNIP表达情况。随后，使用zeste基因增强子同源物2（enhancer of zeste homolog 2, EZH2）甲基转移酶活性抑制剂GSK126处理恶性CTCL细胞系，或采用亚硫酸氢盐测序（bisulphite sequencing）检测TXNIP启动子的甲基化水平；同时使用去甲基化试剂5-氮胞苷（5-azacytidine, 5AZA）评估甲基化状态。最后，通过质粒转导（plasmid transduction）在恶性PB2B细胞系中过表达TXNIP，并借助流式细胞术（flow cytometry）进一步分析TXNIP的生物学效应。 实验结果：相较于非恶性T细胞，本研究发现所有代表不同亚型与分期的CTCL细胞系中，TXNIP蛋白均呈低表达状态。TXNIP的表达抑制涉及表观遗传沉默及其他调控机制，而强制过表达TXNIP可显著抑制恶性T细胞的增殖。 研究结论：恶性T细胞中TXNIP的表达受表观遗传沉默及其他尚未明确的机制所抑制。鉴于强制过表达TXNIP可抑制恶性细胞增殖，我们提出TXNIP是CTCL中潜在的抑癌基因。