NCTD通过靶向Hedgehog/SOX2 axis抑制急性髓系白血病HL60细胞的增殖

NCTD Retards AML HL60 Cell Proliferation via Targeting Hedgehog/SOX2 axis

目的:研究去甲斑蝥素(NCTD)通过抑制Hedgehog信号通路(SHH)关键调节因子及其下游转录因子SOX2对急性髓系白血病细胞增殖的影响。方法:HL60和NB4细胞系经NCTD、SMO和GLI1抑制剂培养24h后,用CCK8检测细胞数目;HL60细胞系经NCTD处理后,利用免疫印迹法检测GLI1、SMO、SOX2的表达情况;将表达GLI1或SOX2的pcDNA3.1质粒转染至HL60细胞,空载体pcDNA3.1和pcDNA 3.1-EGFP分别作为阴性、阳性对照;免疫印迹法检测外源性GLI1、SOX2的表达水平,CCK8检测HL60细胞生长曲线及数目的变化;基因修饰的HL60细胞系经不同浓度NCTD作用后检测HL60细胞增殖情况。结果:NCTD,SMO及GLI1抑制剂以剂量依赖的方式抑制NB4/HL60细胞的增殖。与二甲基亚砜溶剂处理的对照组相比,NCTD可以显著降低SMO、GLI1和SOX2的蛋白水平。与pcDNA3.1空载体转染组相比,GLI1和SOX2在HL60细胞中过表达。生长曲线证实,GLI1/SOX2具有显著增殖优势。CCK8检测表明GLI1/SOX2过表达的HL60细胞对NCTD更具耐药性。结论:NCTD可通过靶向Hedgehog/SOX2 aixs抑制HL60的增殖。

Objective:To investigate the effect of norcantharidin(NCTD)to proliferation of leukemia cells through disrupting key regulators of sonic Hedgehog(SHH)pathway and its downstream transcription factor SOX2.Methods:CCK8 was used to detected the HL60 and NB4 cells after inhibited by NCTD,SMO and GLI1 inhibitor for 24 hours.Expression level of SMO,GLI1 and SOX2 in HL60 cells with NCTD treatment was detected by immunoblot.HL60 cells were transfected with pcDNA3.1 plasmid expressing GLI1 or SOX2.Empty vector and pcDNA3.1-EGFP were divided into negative and positive control group,respectively.The expression of exogenous GLI1 or SOX2 in HL60 cells was confirmed by immunoblot,and growth curve of HL60 cell was checked by CCK8.Proliferation of genetic modified HL60 cells treated by various dose of NCTD was detected.Results:NCTD,SMO/GLI1 inhibitors could inhibit the proliferation of NB4 and HL60 cells in a dose-dependent manner.Compared with solvent(DMSO)-treated control group,NCTD remarkably decreased protein level of SMO,GLI1 and SOX2.GLI1 and SOX2 were overexpressed in HL60 cells as compared with pcDNA3.1 empty vector-transfected group.Growth curve demonstrated significant proliferative advantage of GLI1/SOX2-transfected cells.CCK8 assay indicated that GLI1/SOX2-overexpressed HL60 cells were more resistant to NCTD treatment.Conclusion:NCTD attenuates HL60 proliferation via targeting the Hedgehog/SOX2 axis.