Yan Jian-Guo, Fu Hai-Ying, Shen Jian-Zhen, Zhou Hua-Rong, Zhang Yuan-Yuan, Huang Jing-Long, Chen Cong-Jie, Huang Si-Han
Fujian Provincial Insitute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian Medical University Union Hospital, Fuzhou 350001, Fujian Province,China.
Fujian Provincial Insitute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian Medical University Union Hospital, Fuzhou 350001, Fujian Province,China. E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2016 Oct;24(5):1299-1304. doi: 10.7534/j.issn.1009-2137.2016.05.003.
To detect the abnormal methylation of the CPG island in the suppressor gene promoter region of the Wnt signaling pathway in cell strain NB4 of the acute promyelocytic leukemia by using the bisulfite sequcucing PCR(BSP), to screan the hyper-methylated suppressor gene of the Wnt signaling pathway and to evaluatc the potency of BSP in the methylation study.
The strain NB4 cells of the acute promyelocytic leukemia patients were used as the object, the mononuclear cells from 20 normal persons were used as the controls. The DNA was extracted and processed by bisulfite, the target sequences were amplified with PCR, then the abnormal methylation of the suppressor genes of the Wnt signaling pathway in the NB4 cells was analyzed by BSP, and the advantage and disadvantage of BSP were evaluated by comparison with the Methylation specific PCR and Pyrosequencing.
The methylated rate of suppressor genes of the Wnt signaling pathways in the NB4 cells detected by BSP was as follows: the gene WIF1 95.26%, the gene DKK3 86%, the gene SFRP1 81.67%, the gene SFRP2 95.71%, the gene SFRP4 85%, and the gene SFRP5 95%; while the methylations in the control group were respectively as follows: the gene WIF-1 1.5%, the gene DKK3 4.2%, the gene SFRP1 0%, the gene SFRP2 0.9%, the gene SFRP4 2.5%, and the gene SFRP5 1.75%. A more significant methylation happened in the suppressor genes promoter of the Wnt signaling pathway in the NB4 cells as compared with the control group.
Many hypermethylated suppressor genes are found in the Wnt signaling pathway of the acute promyelocytic leukemia NB4 cells, which may be served as one of the early diagnosis index and therapeutic target of the acute promyelocytic leukemia.
应用亚硫酸氢盐测序PCR（BSP）技术检测急性早幼粒细胞白血病细胞株NB4中Wnt信号通路抑制基因启动子区域CPG岛的异常甲基化，筛选Wnt信号通路高甲基化抑制基因，评估BSP在甲基化研究中的效能。
以急性早幼粒细胞白血病患者的NB4细胞株为研究对象，20名正常人的单个核细胞为对照。提取DNA并经亚硫酸氢盐处理，用PCR扩增目的序列，然后用BSP分析NB4细胞中Wnt信号通路抑制基因的异常甲基化情况，并与甲基化特异性PCR和焦磷酸测序法比较，评估BSP的优缺点。
BSP检测NB4细胞中Wnt信号通路抑制基因的甲基化率分别为：WIF1基因95.26%，DKK3基因86%，SFRP1基因81.67%，SFRP2基因95.71%，SFRP4基因85%，SFRP5基因95%；而对照组中各基因的甲基化率分别为：WIF-1基因1.5%，DKK3基因4.2%，SFRP1基因0%，SFRP2基因0.9%，SFRP4基因2.5%，SFRP5基因1.75%。与对照组相比，NB4细胞中Wnt信号通路抑制基因启动子区甲基化更明显。
急性早幼粒细胞白血病NB4细胞的Wnt信号通路中发现多个高甲基化抑制基因，可能作为急性早幼粒细胞白血病的早期诊断指标和治疗靶点之一。
