Hatırnaz Ng Özden, Fırtına Sinem, Can İsmail, Karakaş Zeynep, Ağaoğlu Leyla, Doğru Ömer, Celkan Tiraje, Akçay Arzu, Yıldırmak Yıldız, Timur Çetin, Özbek Uğur, Sayitoğlu Müge
İstanbul University Faculty of Medicine, Institute of Experimental Medicine Research (DETAE), Department of Genetics, İstanbul, Turkey Phone: +90 212 414 20 00-33312 E-mail:
Turk J Haematol. 2015 Jun;32(2):127-35. doi: 10.4274/tjh.2013.0296.
WNT5A is one of the most studied noncanonical WNT ligands and is shown to be deregulated in different tumor types. Our aim was to clarify whether hypermethylation might be the cause of low WNT5A mRNA levels and whether we could restore this downregulation by reversing the event.
The expression of WNT5A mRNA was studied in a large acute lymphoblastic leukemia (ALL) patient group (n=86) by quantitative real-time PCR. The methylation status was detected by methylation-specific PCR (MSPCR) and bisulphate sequencing. In order to determine whether methylation has a direct effect on WNT5A expression, disease-representative cell lines were treated by 5'-aza-20-deoxycytidine.
Here we designed a validation experiment of the WNT5A gene, which was previously examined and found to be differentially expressed by microarray study in 31 T-cell ALL patients. The expression levels were confirmed by quantitative real-time PCR and the expression levels were significantly lower in T-cell ALL patients than in control thymic subsets (p=0.007). MSPCR revealed that 86% of the patients were hypermethylated in the WNT5A promoter region. Jurkat and RPMI cell lines were treated with 5'-aza-20-deoxycytidine and WNT5A mRNA expression was restored after treatment.
According to our results, WNT5A hypermethylation does occur in ALL patients and it has a direct effect on mRNA expression. Our findings show that epigenetic changes of WNT signaling can play a role in ALL pathogenesis and reversing methylation might be useful as a possible treatment of leukemia.
WNT5A是研究最多的非经典WNT配体之一，在不同肿瘤类型中显示出调控异常。我们的目的是阐明高甲基化是否可能是WNT5A mRNA水平低的原因，以及我们是否可以通过逆转该事件来恢复这种下调。
通过定量实时PCR研究了一大组急性淋巴细胞白血病（ALL）患者（n = 86）中WNT5A mRNA的表达。通过甲基化特异性PCR（MSPCR）和亚硫酸氢盐测序检测甲基化状态。为了确定甲基化是否对WNT5A表达有直接影响，用5'-氮杂-2'-脱氧胞苷处理具有疾病代表性的细胞系。
在此我们设计了WNT5A基因的验证实验，该基因先前已在31例T细胞ALL患者中通过微阵列研究进行检测并发现其表达存在差异。通过定量实时PCR证实了表达水平，T细胞ALL患者的表达水平明显低于对照胸腺亚群（p = 0.007）。MSPCR显示86%的患者在WNT5A启动子区域发生高甲基化。用5'-氮杂-2'-脱氧胞苷处理Jurkat和RPMI细胞系，处理后WNT5A mRNA表达得以恢复。
根据我们的结果，ALL患者中确实发生了WNT5A高甲基化，并且它对mRNA表达有直接影响。我们的研究结果表明，WNT信号通路的表观遗传变化可能在ALL发病机制中起作用，逆转甲基化可能作为白血病的一种可能治疗方法。
