[慢性髓性白血病疾病进展中的SHP-1基因]

[SHP-1 gene in the disease progression of chronic myeloid leukemia].

作者信息

Li Yinghua, Wang Xingzhe, Yang Lin, Pan Yuxia, Shang Yintao, Luo Jianmin

机构信息

Department of Hematology, the Second Hospital of Hebei Medical University, Key Laboratory of Hematology, Hebei Province, Shijiazhuang 050000, China.

出版信息

Zhonghua Xue Ye Xue Za Zhi. 2014 Dec;35(12):1074-8. doi: 10.3760/cma.j.issn.0253-2727.2014.12.006.

DOI:10.3760/cma.j.issn.0253-2727.2014.12.006

PMID:25543700

Abstract

OBJECTIVE

To investigate the profile of promoter methylation and expression of SHP-1 gene in the progression of chronic myeloid leukemia (CML).

METHODS

The expression level of SHP-1 mRNA and protein in bone marrow or peripheral blood mononuclear cells from CML patients were detected by Western blot and SYBR Green-based qRT-PCR. The methylation status of SHP-1 were assessed by methylation-specific polymerase chain reaction (MSP) assay. K562 cells were infected with the lentiviral plasmids pEX-SHP-1-puro-Lv105 (K562-SHP-1) or pEX-EGFP-puro-Lv105 (K562-EGFP). The levels of proteins and phosphorylated proteins were detected by Western blot. qRT-PCR assay was used to test the level of BCR-ABL mRNA.

RESULTS

The relative levels of SHP-1 mRNA were sharply decreased in advanced stages CML compared to chronic phase (CP)-CML (0.79±0.37 vs 1.18±0.64, P= 0.009). The level of SHP-1 protein was lower in advanced stages CML compared to CP-CML (0.57±0.02 vs 1.02±0.04, P=0.039). The frequency of SHP-1 gene promoter methylation at selected loci in CP-CML was 23.8% (10/42), and the methylated regions were detected in all advanced CML samples (P<0.01). SHP-1 was stably transfected into K562 cells and selected with puromycin. Overexpression of SHP-1 inhibited the proliferation and induced the apoptosis of K562 cells, meanwhile leaded to G0/G1 phase arrest. After transfection, the level of BCR-ABL mRNA was not affected in K562-SHP-1 cells (1.32±0.34) compared to K562-EGFP cells (1.18±0.20, P=0.644), but overexpression of SHP-1 caused a slight decrease in BCR-ABL protein in K562-SHP-1 cells compared to K562 -EGFP cells (0.78±0.15 vs 1.27±0.24, P=0.040). Overexpression of SHP-1 resulted in a remarkable decrease in MYC protein, phosphorylated forms of JAK2, STAT5, Akt and MAPK. However, the un-phosphorylated forms of these molecules were not significantly affected.

CONCLUSION

Decreased expression of SHP-1 caused by aberrant promoter hypermethylation may play a key role in the progression of CML by dysregulation of BCR-ABL, Akt, MAPK, MYC, JAK2 and STAT5 signaling.

摘要

目的

探讨慢性髓性白血病（CML）进展过程中SHP-1基因启动子甲基化及表达情况。

方法

采用蛋白质免疫印迹法和基于SYBR Green的定量逆转录-聚合酶链反应（qRT-PCR）检测CML患者骨髓或外周血单个核细胞中SHP-1 mRNA和蛋白的表达水平。通过甲基化特异性聚合酶链反应（MSP）分析SHP-1的甲基化状态。用慢病毒质粒pEX-SHP-1-puro-Lv105（K562-SHP-1）或pEX-EGFP-puro-Lv105（K562-EGFP）感染K562细胞。采用蛋白质免疫印迹法检测蛋白及磷酸化蛋白水平。用qRT-PCR检测BCR-ABL mRNA水平。

结果

与慢性期（CP）-CML相比，晚期CML中SHP-1 mRNA的相对水平显著降低（0.79±0.37 vs 1.18±0.64，P = 0.009）。与CP-CML相比，晚期CML中SHP-1蛋白水平较低（0.57±0.02 vs 1.02±0.04，P = 0.039）。CP-CML中所选位点的SHP-1基因启动子甲基化频率为23.8%（10/42），所有晚期CML样本均检测到甲基化区域（P<0.01）。将SHP-稳定转染入K562细胞并用嘌呤霉素筛选。SHP-1的过表达抑制了K562细胞的增殖并诱导其凋亡，同时导致细胞停滞于G0/G1期。转染后，与K562-EGFP细胞（1.18±0.20）相比，K562-SHP-1细胞中BCR-ABL mRNA水平未受影响（1.32±0.34，P = 0.644），但与K562-EGFP细胞相比，SHP-1的过表达使K562-SHP-1细胞中BCR-ABL蛋白略有下降（0.78±0.15 vs 1.27±0.24，P = 0.040）。SHP-1的过表达导致MYC蛋白、JAK2、STAT5、Akt和MAPK的磷酸化形式显著降低。然而，这些分子的未磷酸化形式未受到显著影响。