摘要
目的探讨神经鞘磷脂合成酶2(SMS2)基因沉默对人乳腺癌细胞MCF-7凋亡的影响以及机制分析。方法体外培养MCF-7细胞,分别转染siRNA control(SiR-Con)和SMS2 siRNA(SiR2),用MTT法筛选肿瘤坏死因子α(TNF-α)诱导MCF-7细胞凋亡的剂量(IC50)、采用薄层层析法(TLC)检测SMS2基因沉默后MCF-7细胞内SMS酶活性水平并用Hoechst 33258/PI染色法检测细胞凋亡情况。采用qRT-PCR和Western blot检测B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)和caspase-3 mRNA和蛋白表达的变化。结果随着TNF-α(50～800 ng/mL)浓度的增加,对MCF-7细胞增殖抑制率也逐渐增加,IC50为205.7 ng/mL。经TNF-α作用后,TNF-α组、SiR-Con组和SiR2组MCF-7细胞内SMS酶活性灰度值明显低于CTL组,且细胞凋亡率和坏死率均高于CTL组;以SiR2组细胞SMS酶活性为最低,凋亡率和坏死率为最高(P <0.05)。SiR2组细胞Bcl-2表达量低于其他3组,而Bax和caspase-3基因表达则高于其他3组(P <0.05)。结论 SMS2基因沉默可降低MCF-7细胞内SMS酶活性,通过抑制抗凋亡基因Bcl-2的表达,同时上调促凋亡基因Bax和caspase-3的表达,提高TNF-α诱导肿瘤细胞凋亡的作用。
Objective To investigate the roles and the impossible mechanisms of sphingomyelin synthase2(SMS2)gene silencing on apoptosis of breast cancer MCF-7 cells.Methods MCF-7 cells were cultured in vitro and transfected with SMS2 siRNA lentivirus vector and negative control lentivirus vector.IC50 of tumor necrosis factorα(TNF-α)of inhibited MCF-7 cells was detected by MTT assay.The SMS activity of MCF-7 cells was detected by thin layer chromatography(TLC).The apoptosis of MCF-7 cells was detected by flow cytometry and Hoechst33258/PI staining.The B-cell lymphoma-2(Bcl-2),bcl-2-associated X protein(Bax)and caspase-3 expressions of MCF-7 cells were detected by qRT-PCR and western blot.Results The IC50 concentration of TNF-α(50~800 ng/mL)on inhibition of MCF-7 cells was 205.7 ng/mL.After treated with TNF-α,the gray value of SMS activity in MCF-7 cells in TNF-αgroup,SiR-Con group and SiR2 group was significantly lower than that in CTL group,and the apoptosis rate and necrosis rate were higher than those in CTL group.In SiR2 group the activity of SMS was the lowest,and the apoptosis rate and necrosis rate were the highest(P<0.05).The expression of Bcl-2 mRNA and protein in MCF-7 cells with siSMS2 were lower than the other 3 groups(P<0.05),whereas the expressions of Bax and caspase-3 in SiR2 group were higher than the other 3 groups(P<0.05).Conclusion There are significant evidences that SMS2 gene silencing would decrease SMS activity of MCF-7 cells and enhance the effect of TNF-αinducing the apoptosis of MCF-7 cells by down-regulation of Bcl-2 and up-regulation of Bax and caspase-3.
作者
杭月
林昌岫
黄成日
HANG Yue;LIN Changxiu;HUANG Chengri(Department of Gynaecology and Obstetrics,Affiliated Hospital of Yanbian University,Yanji 133000,China)
出处
《实用医学杂志》
CAS
北大核心
2019年第8期1227-1232,共6页
The Journal of Practical Medicine
基金
国家自然科学基金项目(编号:81360382)
吉林省教育厅"十三五"科学技术项目(编号:JJKH20191118KJ)
