Epigenetic Silencing of TFPI-2 in Canine Diffuse Large B-Cell Lymphoma

Epigenetic modifications are important early events during carcinogenesis. In particular, hypermethylation of CpG islands in the promoter region of tumor suppressor genes is a well-known mechanism of gene silencing that contributes to cancer development and progression. Tissue factor pathway inhibitor 2 (TFPI-2) is a tumor suppressor involved in invasiveness inhibition. Although TFPI-2 transcriptional silencing, through promoter hypermethylation, has been widely reported in several human malignancies, it has never been explored in lymphoma. In the present study TFPI-2 methylation and gene expression have been investigated in canine Diffuse Large B-cell lymphomas (cDLBCL). The methylation level of 23 CpGs located within the TFPI-2 promoter was investigated by bisulfite-specific PCR and next generation amplicon deep sequencing (GS Junior 454, Roche) in 22 cDLBCLs and 9 controls. For the same specimens, TFPI-2 gene expression was assessed by means of Real-time RT-PCR. Sequence analysis clearly demonstrated that TFPI2 is frequently hypermethylated in cDLBCL. Hypermethylation of the TFPI-2 promoter was found in 77% of DLBCLs (17 out of 22) and in one normal lymph node. Globally, dogs with DLBCL showed a mean methylation level significantly increased compared to controls (p<0.01) and analysis of hypermethylation by site identified 19 loci out of 23 (82%) with mean methylation levels from 2- to 120-fold higher in cDLBCL. Gene expression analysis confirmed a significant down-regulation of TFPI-2 (p<0.05) in DLBCLs compared with normal lymph nodes, suggesting that TFPI-2 hypermethylation negatively regulates its transcription. In addition, a significant positive correlation (p<0.01) was found between TFPI-2 methylation levels and age providing the first indication of age-associated epigenetic modifications in canine DLBCL. To conclude, our findings demonstrated that epigenetic dysregulation of TFPI-2, leading to its reduced expression, is frequently detected in canine DLBCL. In the next future, the aberrant TFPI-2 promoter hypermethylation may be considered in association with prognosis and therapy.表观遗传修饰（epigenetic modifications）是致癌过程中的重要早期事件。具体而言，抑癌基因启动子区域内CpG岛（CpG islands）的高甲基化是一种公认的基因沉默机制，可促进癌症的发生与发展。组织因子途径抑制物2（Tissue factor pathway inhibitor 2, TFPI-2）是一种可抑制肿瘤侵袭的抑癌基因。尽管通过启动子高甲基化实现的TFPI-2转录沉默已在多种人类恶性肿瘤中被广泛报道，但目前尚未有其在淋巴瘤中相关研究的报道。 本研究针对犬弥漫大B细胞淋巴瘤（canine Diffuse Large B-cell lymphomas, cDLBCL）展开TFPI-2甲基化水平与基因表达的相关研究。研究采用亚硫酸氢盐特异性PCR（bisulfite-specific PCR）与下一代扩增子深度测序（next generation amplicon deep sequencing，罗氏GS Junior 454平台），对22例cDLBCL样本与9例对照样本中TFPI-2启动子区域内23个CpG位点的甲基化水平进行检测；同时针对上述同一批样本，采用实时荧光定量逆转录聚合酶链反应（Real-time RT-PCR）对TFPI-2的基因表达水平进行检测。 序列分析结果明确显示，TFPI-2在cDLBCL中频繁出现高甲基化现象。TFPI-2启动子的高甲基化在77%的DLBCL样本（22例中的17例）以及1例正常淋巴结样本中被检测到。整体而言，与对照组相比，DLBCL患犬的平均甲基化水平显著升高（p<0.01）；位点特异性高甲基化分析显示，23个位点中有19个（占比82%）在cDLBCL中的平均甲基化水平较对照组升高2至120倍。 基因表达分析证实，与正常淋巴结样本相比，DLBCL样本中TFPI-2的表达显著下调（p<0.05），这表明TFPI-2高甲基化可对其转录产生负向调控作用。此外，本研究还发现TFPI-2甲基化水平与患犬年龄存在显著正相关（p<0.01），这是首次在犬弥漫大B细胞淋巴瘤中发现与年龄相关的表观遗传修饰改变。 综上，本研究结果证实，TFPI-2的表观遗传调控异常（导致其表达水平降低）在犬弥漫大B细胞淋巴瘤中频繁出现。未来可将TFPI-2启动子的异常高甲基化作为该疾病预后评估与治疗方案制定的潜在关联指标。