SFRP5基因甲基化通过激活Wnt/β-catenin通路调节白血病细胞MDR1/P-gp的表达

Methylation of SFRP5gene regulates MDR1/P-glycoprotein expression in leukemic cells through Wnt/β-catenin pathway

目的:研究分泌型卷曲相关蛋白5(SFRP5)基因甲基化对耐药白血病细胞多药耐药蛋白1/P-糖蛋白(MDR1/P-gp)表达的调节及其可能的信号通路。方法:采用甲基化特异性PCR(MSP)检测白血病患者及白血病细胞系中SFRP5基因启动子区甲基化状态。采用Western blotting检测非磷酸化β-catenin(NP-β-catenin)在SFRP5基因甲基化白血病患者及细胞系中表达。使用去甲基化试剂5-氮杂-2'-脱氧胞嘧啶(DAC)恢复SFRP5表达,采用RT-PCR和real-time PCR检测处理前后MDR1 mRNA表达,采用Western blotting检测P-gp表达。结果:5/12例白血病患者标本及4种白血病细胞系存在SFRP5基因完全甲基化。在5例患者标本中,有3例(L2、L7和L8)NP-β-catenin水平高于其它标本,在4种白血病细胞系中,KG1a中NP-β-catenin水平高于另外3个细胞系。存在NP-β-catenin高水平表达的3例患者(L2、L7和L8)中检出MDR1 mRNA及P-gp表达。在4种细胞系中,KG1a存在MDR1 mRNA及P-gp表达。使用去甲基化试剂DAC处理L2、L7、L8患者标本和KG1a细胞,恢复SFRP5表达,L2、L7、L8和KG1a中MDR1 mRNA水平均显著下降(P<0.01),P-gp表达水平亦被下调。结论:SFRP5基因甲基化可导致Wnt/β-catenin信号通路被激活,活化的β-catenin激活下游靶基因MDR1转录,编码的药物外排泵P-gp表达增加,促进白血病多药耐药的形成。

AIM： To investigate the role of secreted frizzled-related protein 5 （SFRP5） gene methylation in the expression of muhidrug resistance 1/P-glycoprotein （MDR1/P-gp） in leukemic cells and to explore the possible signa- ling pathway. METHODS ： The methylation status of SFRP5 gene promoter region in leukemic patients and cell lines was detected by methylation-specific PCR （MSP）. Non-phosphorylated ^-catenin （NP-tS-catenin） expression in the leukemic patients and the cell lines was also detected by Western blotting. The demethylation reagent 5-aza-2＇-deoxycytidine （DAC） was used to recover SFRP5 expression in P-gp positive specimens. The level of P-gp was determined by Western blotting, and the mRNA expression of MDR1 was measured by RT-PCR and real-time PCR. RESULTS： Complete methylation of SFRP5 gene was observed in 5 cases of total 12 leukemic patients and the cell lines HL-60, Raji, U937 and KGla. The expression levels of NP-13-catenin in 3 cases （ L2, L7 and L8 ） were higher than the others. The expression of NP-13-catenin in KGla cells was significantly higher than that in other leukemic cell lines. The mRNA expression of MDRI and the pro- tein level of P-gp were detected in the 3 patients who had higher levels of NP-13-catenin. Among the 4 cell lines, KGla cells positively expressed MDR1 mRNA and P-gp protein. Restoration of SFRP5 expression by treatment with demethylation reagent DAC down-regulated the protein levels of P-gp and significantly decreased the mRNA expression of MDR1 in L2, L7 and L8 patients and KG1 a cells. CONCLUSION： SFRP5 gene promoter is methylated in leukemic cells, leading to activate the Wnt/β-catenin signaling pathway, and increase the transcription of MDR1 gene and expression of drug efflux pump P-gp, thus promoting the formation of multidrug resistance.