布托啡诺通过下调布托啡诺调节音猬因子/胶质瘤相关癌基因同源物1通路抑制胶质瘤细胞恶性生物学行为

Butorphanol inhibits malignant biological behavior of glioma cells by down-regulating the sonic hedgehog/glioma-associated oncogene homolog 1 pathway

目的探讨布托啡诺调节音猬因子(SHH)/胶质瘤相关癌基因同源物1(GLI1)信号通路对胶质瘤细胞恶性生物学行为的影响。方法将胶质瘤LN229细胞分为对照组、布托啡诺低剂量组、布托啡诺中剂量组、布托啡诺高剂量组以及布托啡诺高剂量+pc DNA3.1组、布托啡诺高剂量+pc-SHH组。MTT和Edu实验检测细胞增殖能力;流式细胞术检测细胞凋亡率;Transwell小室实验检测细胞迁移和侵袭;实时荧光定量聚合酶链反应(qRT-PCR)检测SHH mRNA、GLI1 mRNA表达水平;蛋白免疫印迹(Western blot)检测Ki-67、基质金属蛋白酶-2(MMP-2)、裂解的半胱天冬酶-3(Cleaved-Caspase-3)、SHH和GLI1蛋白表达水平。结果布托啡诺可降低LN229细胞活力、增殖率,减少细胞迁移和侵袭个数(P<0.05)。布托啡诺可降低LN229细胞中SHH mRNA、GLI1 mRNA表达水平及Ki-67、MMP-2、SHH和GLI1蛋白表达,促进细胞凋亡和增高Cleaved-Caspase-3蛋白表达(P<0.05)。过表达SHH可部分逆转布托啡诺对LN229细胞的抑制作用(P<0.05)。结论布托啡诺可以抑制胶质瘤细胞的恶性生物学行为,其机制可能与抑制SHH/GLI1信号通路有关。

Objective To investigate the effect of butorphanol on the malignant biological behavior of glioma cells by regulating the sonic hedgehog(SHH)/glioma-associated oncogene homolog 1(GLI1)signaling pathway.Methods Glioma LN229 cells were divided into control group,low-dose butorphanol group,medium-dose butorphanol group,high-dose butorphanol group,high-dose butorphanol+pcDNA3.1 group and high-dose butorphanol+pc-SHH group.MTT and Edu assays were used to detect cell proliferation;flow cytometry was used to detect cell apoptosis rate;Transwell chamber assay was used to detect cell migration and invasion;real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)was used to detect SHH mRNA and GLI1 mRNA expression levels;Western blot was used to detect the protein expression levels of Ki-67,matrix metalloproteinase-2(MMP-2),cleaved caspase-3,SHH and GLI1.Results Butorphanol could reduce the viability and proliferation rate of LN229 cells,and decreased the number of cell migration and invasion(P<0.05).Butorphanol could reduce the expression levels of SHH mRNA,GLI1 mRNA and the protein expression of Ki-67,MMP-2,SHH as well as GLI1 in LN229 cells,promote cell apoptosis and increase the protein expression of cleaved caspase-3(P<0.05).Overexpression of SHH could partially reverse the inhibitory effect of butorphanol on LN229 cells(P<0.05).Conclusion Butorphanol can inhibit the malignant biological behavior of glioma cells,and its mechanism may be related to the inhibition of the SHH/GLI1 signaling pathway.