萝卜硫素预防Ⅰ型神经纤维瘤病恶性进展的作用机制研究

he effect and mechanism of sulforaphane in preventing malignant progression of neurofibromatosis typeⅠ

目的:研究萝卜硫素(SFN)预防Ⅰ型神经纤维瘤(NF-1)进展为恶性周围神经鞘瘤(MPNST)的作用及有关机制。方法:使用CCK-8、克隆形成实验检测细胞增殖能力;尼罗红(Nile Red)染色法检测细胞内脂肪含量;CellTiter-Glo 2.0 Assay试剂盒检测细胞内ATP含量;采用实时荧光定量PCR检测细胞脂肪酸合酶(FASN)、乙酰辅酶A羧化酶1(ACC1)、肉碱棕榈酰转移酶1A(CPT1A)的mRNA水平,免疫印迹实验检测细胞β-catenin、Axin 2、C-Myc、Cyclin D1的蛋白水平。结果:SFN抑制STS26T、ST88-14细胞增殖活性(t=18.70、11.20,均P<0.05),减少细胞克隆数量(t=7.28、3.148,均P<0.05),并减少细胞内脂质积累(t=3.411、5.75,均P<0.05)和ATP的含量(t=14.75、13.53,均P<0.05);与对照组相比,SFN降低STS26T和ST88-14细胞中FASN和CPT1A的mRNA水平(均P<0.05);与对照组相比,15μmol/L SFN可下调STS26T和ST88-14细胞内β-catenin及下游靶基因Axin 2、C-Myc、Cyclin D1的蛋白表达水平(均P<0.05)。结论:SFN能够通过抑制细胞脂肪酸合成及β-氧化和抑制Wnt/β-catenin信号通路来抑制MPNST细胞增殖,具有预防NF-1恶性转化为MPNST的潜力。

Objective:To investigate the effect and mechanism of sulforaphane(SFN)in preventing the progression of malignant peripheral nerve sheath tumors(MPNST)in neurofibromatosis type 1(NF-1).Methods:CCK-8 and colony formation assay were used to detect cell proliferation.Nile Red staining was used to detect intracellular fat content.CellTiter-Glo 2.0 Assay kit was used to detect intracellular triphosadenine(ATP)content.The mRNA levels of fatty acid synthase(FASN),acetyl-CoA carboxylase 1(ACC1),and carnitine palmitoyltransferase 1A(CPT1A)were detected by real-time fluorescence quantitative PCR.The protein levels ofβ-catenin,Axin 2,C-Myc,and Cyclin D1 were detected by Western blotting.Results:SFN inhibited the proliferation of STS26T and ST88-14 cells(t=18.70,11.20,both P<0.05),reduced the number of cell clones(t=7.28,3.148,both P<0.05),a reduced intracellular lipid accumulation(t=3.411,5.75,both P<0.05)and ATP content(t=14.75,13.53,both P<0.05).Compared with the control group,SFN reduced the mRNA levels of FASN and CPT1A in STS26T and ST88-14 cells(both P<0.05).Compared with the control group,15μmol/L SFN down-regulated the protein expression levels ofβ-catenin and its target genes Axin 2,C-Myc and Cyclin D1(all P<0.05).Conclusion:Sulforaphane can inhibit the proliferation of MPNST cells by inhibiting fatty acid synthesis,β-oxidation and Wnt/β-catenin signaling pathway,and has the potential to prevent the malignant transformation of NF-1 to MPNST.