摘要
目的分析短发夹RNA(shRNA)介导DEAD⁃box解螺旋酶46(DDX46)基因沉默对乳腺癌细胞增殖和凋亡的影响。方法采用实时荧光定量逆转录PCR(RT⁃qPCR)和蛋白质印迹法(Western Blot)测定人正常乳腺上皮细胞系和乳腺癌细胞系中DDX46表达差异。以慢病毒介导的shRNA敲低乳腺癌SK⁃BR⁃3细胞中DDX46的表达,实验分为空白对照组、阴性对照组和sh⁃DDX46组。RT⁃qPCR检测各组细胞DDX46 mRNA的表达水平,蛋白质印迹法检测DDX46蛋白和凋亡相关蛋白的表达水平,克隆形成和四甲基偶氮唑盐微量酶反应比色法(MTT法)检测细胞生长增殖,流式细胞仪检测细胞凋亡。结果DDX46 mRNA在各乳腺癌细胞系中的表达量均高于正常乳腺上皮细胞(P<0.05)。sh⁃DDX46组细胞DDX46 mRAN和蛋白表达水平均明显低于空白对照组和阴性对照组(P<0.05)。sh⁃DDX46组的细胞生长增殖能力明显低于两对照组(P<0.05)。空白对照组、阴性对照组和sh⁃DDX46组SK⁃BR⁃3细胞凋亡率分别为(4.21±1.65)%、(5.36±1.23)%和(10.21±2.39)%,与空白对照组和阴性对照组相比,sh⁃DDX46组SK⁃BR⁃3细胞凋亡率明显增加(P=0.007;P=0.016)。沉默DDX46后凋亡通路蛋白B细胞淋巴瘤/白血病⁃2(Bcl⁃2)的表达量明显降低(P<0.05),Bcl⁃2相关X蛋白(Bax)和半胱氨酸蛋白酶⁃3剪切体(cleaved Caspase⁃3)表达明显增高(P<0.05)。结论DDX46在乳腺癌细胞中高表达,沉默DDX46基因能够抑制乳腺癌细胞增殖并促进凋亡,凋亡信号通路可能是其作用机制之一。
Objective To investigate the effects of short hairpin RNA(shRNA)interference targeting RNA helicases DEAD⁃box 46(DDX46)gene silencing on the proliferation and apoptosis of breast cancer cells.Methods The expressions of DDX46 mRNA in different breast cancer cell lines and normal mammary epithelial cell line were determined by real⁃time fluorescent quantitative reverse transcription PCR(RT⁃qPCR)and Western blotting.The breast cancer SK⁃BR⁃3 cells were transfected with DDX46 shRNA(sh⁃DDX46 group)or scrambled sequences(negative control group),with untransfected SK⁃BR⁃3 cells as blank control(blank control group).DDX46 mRNA and protein levels of SK⁃BR⁃3 cells were detected after transfected respectively by RT⁃qPCR and Western blotting.The cell proliferation and apoptosis were measured by colony formation assay,MTT assay and flow cytometry,and the apoptosis⁃related proteins expression levels were detected by Western blot analysis.Results The relative expression levels of DDX46 mRNA in all studied breast cancer cell lines were significantly higher than that in normal mammary epithelial cells(P<0.05).DDX46 mRNA and protein expressions in sh⁃DDX46 group were significantly lower than those in blank control group and negative control group(P<0.05).Cell growth and proliferation in sh⁃DDX46 group were significantly lower than those in the other two groups(P<0.05).The apoptosis rates of SK⁃BR⁃3 cells in blank control group,negative control group and sh⁃DDX46 group were(4.21±1.65)%,(5.36±1.23)%and(10.21±2.39)%,respectively.Compared with blank control group and negative control group,the apoptosis rate of SK⁃BR⁃3 cells in sh⁃DDX46 group was significantly increased(P=0.007;P=0.016).After DDX46 shRNA transfection,the expression levels of B⁃cell lymphoma/Leukemia⁃2(Bcl⁃2)significantly decreased(P<0.05),and the ex⁃pression of Bcl⁃2 associated X protein Bax and cleaved Caspase⁃3 significantly increased(P<0.05).Conclusion DDX46 is over⁃expressed in breast cancer cells.DDX46 gene
作者
张杨蕊
ZHANG Yangrui(Department of Pathology,The First Affiliated Hospital of Zhengzhou University,Zhengzhou,Henan 450000,China)
出处
《安徽医药》
CAS
2020年第12期2489-2493,I0005,共6页
Anhui Medical and Pharmaceutical Journal
