Objective: To study the function of ten-eleven translocation 2 (Tet2) in γ globin gene expression in patients with β- thalassemia. Methods: Gamma globin expression was induced by 5-azacytidine and Tet2 gene expression was knocked down by short hairpin RNA (shRNA) in a human immortalized myelogenous leukemia K562 cell line. The global 5-hydroxymethylcytosine (5hmC) level was measured by an ELISA kit. 5hmC level of γ globin gene was quantified by sulfite sequencing. The mRNA level of Tet2, γ globin, and related transcription factors Nfe4 and Klf1 were quantified by real-time PCR. Results: Tet2 knockdown resulted in a decreased global 5hmC level from 0.14% to 0.03% as of the control group in K562 cells. The expression of γ globin was enhanced after 5-azacytidine treatment in vitro. However, γ globin mRNA level in Tet2 knockdown cells was only 55% as that in control group. The CG sites on γ globin gene were unmethylated. As Tet2 was down-regulated, the expression levels of Nfe4 and Klf1 decreased by about 80% and increased to 3.5 folds, respectively. Conclusions: Tet2 appears to maintain 5hmC level and facilitates γ globin gene activation. Moreover, Tet2 more likely regulates γ globin expression via affecting transcription factors rather than the gene itself. Thus, Tet2 could be a potential therapeutic target for β thalassemias.
目的： 为提高诱导γ珠蛋白（γ globin）基因激活的效率，研究DNA羟甲基化酶（DNA双加氧酶）基因Tet2在诱导γ珠蛋白激活表达过程中的作用，为β地中海贫血病的治疗提供理论依据。 方法： 在人红白血病细胞系K562中用短发夹RSNA（shRNA）干扰的方法下调Tet2表达后，用酶联免疫吸附测定（ELISA）法检测干扰后细胞基因组5-羟甲基胞嘧啶（5hmC）水平的变化，并用亚硫酸盐测序法检测γ珠蛋白上CG位点羟甲基化状态，最后用荧光定量实时PCR检测药物5-氮胞苷诱导γ珠蛋白激活表达量和γ珠蛋白相关转录因子Nfe4和Klf1表达量的变化。 结果： 不做处理的对照组K562细胞基因组5hmC水平为0.14%，sh-Tet2组（Tet2下调后）约为0.03%；被5-氮胞苷激活后，Tet2下调组（即sh-Tet2+5-氮胞苷组）γ珠蛋白的表达量为未下调组（5-氮胞苷组）的55%左右，但γ珠蛋白上CG位点的羟甲基化状态没有发现明显变化。同时，γ珠蛋白相关转录因子Nfe4和Klf1的表达量变化显著，sh-Tet2组（Tet2下调后）Nfe4为对照组的约20%，Klf1为对照组的3.5倍。 结论： Tet2可维持K562基因组5hmC水平，对γ珠蛋白的激活表达起到一定的调控作用，其作用机制有可能通过调控其上游转录因子来间接发挥调节作用，由此推测Tet2是一个潜在的β地中海贫血的治疗靶点。.
Keywords: 5-Hydroxymethylcytosine; Beta-thalassemia; Tet2; γglobin.