C-myc-siRNA通过调控ROS诱导子宫内膜癌细胞凋亡
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摘要
为探讨原癌基因C-myc-siRNA对子宫内膜癌细胞凋亡的影响,本研究利用细胞计数盒(cell counting Kit-8, CCK-8)法分别检测C-myc-siRNA转染组和空载体转染组(Vector-NC)的子宫内膜癌细胞活力;蛋白免疫印迹法(Western blotting)分别检测C-myc-siRNA转染组和空载体转染组(Vector-NC)的子宫内膜癌细胞凋亡蛋白Bax以及抗凋亡蛋白Bcl-2的变化;流式细胞仪分别检测C-myc-siRNA转染组和空载体转染组(Vector-NC)活性氧簇(reactive oxygen species, ROS)的变化;蛋白免疫印迹法(Western blotting)分别检测预处理NAC (ROS抑制剂)后,C-myc-siRNA转染组和空载体转染组(Vector-NC)的子宫内膜癌细胞凋亡相关蛋白Bax以及Bcl-2的变化。结果表明:C-myc-siRNA转染子宫内膜癌细胞后,促凋亡相关蛋白Bax的表达显著高于空载体转染组,且抗凋亡相关蛋白Bcl-2的表达明显低于空载体转染组(p<0.05);C-myc-siRNA转染子宫内膜癌细胞后,细胞ROS水平明显增加(p<0.05);NAC预处理显著减弱C-myc-siRNA对子宫内膜癌细胞凋亡的促进作用(p<0.05)。本研究结论表明,C-myc-siRNA能够通过调控ROS诱导子宫内膜癌细胞凋亡。
In order to discuss the effects of C-myc-siRNA on the apoptosis of endometrial carcinoma cell. In this research, Cell Counting Kit-8 was used to detect endometrial carcinoma cell viability of C-myc-siRNA-transfected group and Vector-NC group. Western blotting was used to detect the changes of endometrial carcinoma apoptosis protein Bax and anti-apoptosis protein Bcl-2 in C-myc-siRNA transfected group and Vector-NC group. Flow cytometry(FCM) was used to evaluate the changes of reactive oxygen species(ROS) in C-myc-siRNA transfected group and Vector-NC group. Western blot was used to detect the changes of endometrial carcinoma apoptosis related protein Bax and Bcl-2 in C-myc-siRNA transfected group and Vector-NC group after the pre-treatment with NAC(ROS inhibitor). The results revealed that after C-myc-siRNA transfected endometrial carcinoma cell, the expression of pro-apoptotic related protein Bax was significantly higher than that in Vector-NC group and the expression of anti-apoptotic related protein Bcl-2 was significantly lower than that in Vector-NC group(p<0.05). After C-myc-siRNA transfected endometrial carcinoma cell, the ROS level of endometrial carcinoma cell was significantly higher(p<0.05). Pretreatment with NAC significantly weakened the acceleration of C-myc-si RNA on the apoptosis of endometrial carcinoma cell(p<0.05). The conclusion of this study demonstrated that C-myc-siRNA could induce the apoptosis of endometrial carcinoma cell by the regulation of reactive oxygen species.
In order to discuss the effects of C-myc-siRNA on the apoptosis of endometrial carcinoma cell. In this research, Cell Counting Kit-8 was used to detect endometrial carcinoma cell viability of C-myc-siRNA-transfected group and Vector-NC group. Western blotting was used to detect the changes of endometrial carcinoma apoptosis protein Bax and anti-apoptosis protein Bcl-2 in C-myc-siRNA transfected group and Vector-NC group. Flow cytometry(FCM) was used to evaluate the changes of reactive oxygen species(ROS) in C-myc-siRNA transfected group and Vector-NC group. Western blot was used to detect the changes of endometrial carcinoma apoptosis related protein Bax and Bcl-2 in C-myc-siRNA transfected group and Vector-NC group after the pre-treatment with NAC(ROS inhibitor). The results revealed that after C-myc-siRNA transfected endometrial carcinoma cell, the expression of pro-apoptotic related protein Bax was significantly higher than that in Vector-NC group and the expression of anti-apoptotic related protein Bcl-2 was significantly lower than that in Vector-NC group(p<0.05). After C-myc-siRNA transfected endometrial carcinoma cell, the ROS level of endometrial carcinoma cell was significantly higher(p<0.05). Pretreatment with NAC significantly weakened the acceleration of C-myc-si RNA on the apoptosis of endometrial carcinoma cell(p<0.05). The conclusion of this study demonstrated that C-myc-siRNA could induce the apoptosis of endometrial carcinoma cell by the regulation of reactive oxygen species.
引文
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Davis A.C.,Wims M.,Spotts G.D.,Hann S.R.,and Bradley A.,1993,A null C-myc-si RNA mutation causes lethality before 10.5 days of gestation in homozygotes and reduced fertility in heterozygous female mice,Genes&development,1993,7(4):671-682
Hoetelmans R.,van Slooten H.J.,Keijzer R.,Erkeland S.,van de Velde C.J.,and Dierendonck J.H.,2000,Bcl-2 and Bax proteins are present in interphase nuclei of mammalian cells,Cell Death Differ.,7(4):384-392
Kalra N.,and Kumar V.,2004,c-Fos is a mediator of the C-myc-siRNA-induced apoptotic signaling in serum-deprived hepatoma cells via the p38 mitogen-activated protein kinase pathway,J.Biol.Chem.,279(24):25313-25319
Kitson S.,Ryan N.,and Mac Kintosh M.L.,2018,Interventions for weight reduction in obesity to improve survival in women with endometrial cancer,Cochrane Database Syst.Rev.,2:012513
Kloppel G.,and La Rosa S.,2018,Ki67 labeling index:assessment and prognostic role in gastroenteropancreatic neuroendocrine neoplasms,Virchows Arch.,472(3):341-349
Liu J.,and Tao M.F.,2014,Research progress of related genes of endometrial cancer,Journal of Shanghai Jiaotong University(Medical Science),34(3):385-388,403
McAlpine J.,Leon-Castillo A.,and Bosse T.,2018,The rise of a novel classification system for endometrial carcinoma;Integration of Molecular Subclasses,244(5):538-549
Zhang Y.,2011,The relationship between Bcl-2 antisense therapy and tumors,Journal of Modern Oncology,19(2):382-385
Benassi B.,Fanciulli M.,and Fiorentino F.,2006,C-myc-siRNAphosphorylation is required for cellular response to oxidative stress,Molecular Cell,21(4):509-519
Dang C.V.,1999,C-myc-siRNA target genes involved in cell growth,apoptosis,and metabolism,Molecular and Cellular Biology,19(1):1-11
Davis A.C.,Wims M.,Spotts G.D.,Hann S.R.,and Bradley A.,1993,A null C-myc-si RNA mutation causes lethality before 10.5 days of gestation in homozygotes and reduced fertility in heterozygous female mice,Genes&development,1993,7(4):671-682
Hoetelmans R.,van Slooten H.J.,Keijzer R.,Erkeland S.,van de Velde C.J.,and Dierendonck J.H.,2000,Bcl-2 and Bax proteins are present in interphase nuclei of mammalian cells,Cell Death Differ.,7(4):384-392
Kalra N.,and Kumar V.,2004,c-Fos is a mediator of the C-myc-siRNA-induced apoptotic signaling in serum-deprived hepatoma cells via the p38 mitogen-activated protein kinase pathway,J.Biol.Chem.,279(24):25313-25319
Kitson S.,Ryan N.,and Mac Kintosh M.L.,2018,Interventions for weight reduction in obesity to improve survival in women with endometrial cancer,Cochrane Database Syst.Rev.,2:012513
Kloppel G.,and La Rosa S.,2018,Ki67 labeling index:assessment and prognostic role in gastroenteropancreatic neuroendocrine neoplasms,Virchows Arch.,472(3):341-349
Liu J.,and Tao M.F.,2014,Research progress of related genes of endometrial cancer,Journal of Shanghai Jiaotong University(Medical Science),34(3):385-388,403
McAlpine J.,Leon-Castillo A.,and Bosse T.,2018,The rise of a novel classification system for endometrial carcinoma;Integration of Molecular Subclasses,244(5):538-549
Zhang Y.,2011,The relationship between Bcl-2 antisense therapy and tumors,Journal of Modern Oncology,19(2):382-385