骨髓增生异常综合征患者SHP-1基因启动子甲基化状态及相关机制的探讨

Experimental study of SHP-1 promoter methylation in myelodysplastic syndromes and its related mechanism

目的探讨SHP-1基因异常甲基化在骨髓增生异常综合征（MDS）发生、发展中的作用和相关机制。方法将63例MDS患者根据IPSS积分系统分为较低危和较高危组。采用甲基化特异性PCR（MS-PCR）对MDS细胞系SKK-1细胞和MDS患者骨髓细胞进行SHP．1基因启动子甲基化状态的检测；用Westernblot方法检测正常对照者和患者骨髓单个核细胞和SKK-1细胞磷酸化信号传导和转录激活因子3（p-STAT3）蛋白的表达。结果SKK．1细胞存在SHP-1基因启动子部分甲基化，正常对照者骨髓细胞均为非甲基化，较高危组MDS患者SHP-1启动子甲基化率显著高于较低危组（63．3％对24．2％）（P〈0．05）；按WHO分型中染色体核型和骨髓原始细胞比例分组，SHP-1启动子甲基化率在RAEB-Ⅱ、核型差组和骨髓原始细胞占0．11-0．19组显著增高（P〈0．05）；SKK-1细胞中存在p-STAT3蛋白的表达，较高危组MDS患者P-STAT3蛋白的表达显著高于较低危组（66．7％对18．2％）；相关分析显示SHP-1启动子甲基化的出现与p-STAT3蛋白的表达有相关性。结论SHP．1基因启动子的异常甲基化在MDS的发病和疾病进展中发挥作用，其机制可能涉及STAT3信号通路的激活，检测MDS患者SHP-1启动子甲基化状态有助于对疾病预后的判断。

Objective To explore the role of SHP-1 promoter methylation on the pathogenesis and progression in myelodysplastic syndromes （MDS） and its related mechanism. Methods 63 MDS patients were divided into low-grade（LG） group and high-grade （HG） group according to IPSS score system. Bone marrow samples were collected. Methylation specific-PCR （MSP） were used to detect the status of SHP-1 promoter methylation in bone marrow （ BM ） samples from different risk MDS patients and MDS cell line, SKK-1. Western blot was used to detect signal transduction and activator of transcription （STAT3） activation in SKK-1 cell line and MDS patients. Results No SHP-1 promoter methylation could be detected in healthy controls BM. Partially methylation was found in SKK-1 cell line. Methylation rate of SHP-1 gene promoter was found in BM of 24.2% of low-grade MDS patients and 63.3% of high-grade MDS patients, the difference be- tween these two groups was statistically significant （ P 〈 0.05 ） ; Patients were divided into different groups ac- cording to WHO subtype, chromosomal karyotype and blast cells in bone marrow, methylation rates of SHP-1 were significantly higher in RAEB-11 , poor karotype group and samples with 0.11 -0.19 blast cells （P 〈 0.05 ） ;The phosphorylation protein of STAT3 was detected in SKK-1 cell line. The expression of phosphoryla- tion STAT3 was significantly higher in HG group than in LG group （66.7% vs 18.2% ） （P 〈0.05）. There was a significant correlation between SHP-1 promoter methylation and STAT3 phosphorylation. Conclusion Abnormal methylation of SHP-1 gene promoter might have tentative role in the pathogenesis and progression of MDS, which may be involved in STAT3 activation. Detection of SHP-1 promoter methylation may be helpful to evaluate the orognosis of MDS.