去泛素化酶USP9X在etoposide促结肠癌细胞SW620凋亡中的作用研究
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摘要
目的:探讨USP9X在etoposide促结肠癌细胞SW620凋亡中的作用和可能的机制。方法:设计并合成USP9X的shRNA和对照shcontrol,采用半定量RT-PCR和Western blot检测干扰效率。结肠癌细胞SW620分别转染USP9X-shRNA和shcontrol后用etoposide处理,检测MCL1的蛋白水平变化并通过c-PARP的水平检测细胞的凋亡水平。结果:半定量RT-PCR和Western blot检测显示在SW620细胞中USP9X-shRNA能够显著降低USP9X的mRNA和蛋白的表达,抑制率达70%,该条shRNA可以作为有效干扰序列进行后续实验。20μg/mL etoposide处理24 h后,经c-PARP的水平检测发现感染USP9X-shRNA的SW620细胞比对照组细胞的凋亡率显著增加。结论:以RNA干扰技术沉默USP9X基因可增加结肠癌细胞SW620对etoposide的敏感性,显著增加etoposide诱导的结肠癌SW620细胞的凋亡,推测USP9X基因可能成为结肠癌基因治疗的一个新靶点。
Objctives: To investigate the effect of USP9X on apoptosis of colon cancer induced by etoposide and its potential mechanism. Methods: USP9X small hairpin RNA(shRNA)sequence and control shRNA sequence were designed and synthesized, and the interference effective of USP9X- shRNA was detected by semi- quantitative RT- PCR and Western blotting. After transfected with USP9X- shRNA and shcontrol, SW620 cells were treated with etoposide, the de-tection of c- PARP were used to analysis the apoptosis of the cell. Results: Semi- quantitative RT- PCR and Western blot showed that the mRNA and protein level of USP9X were downregulated in SW620 cells transfected with USP9X- shRNA, and the inhibition rate reach to 70%. The shRNA sequence worked well for the following experiments. After treated with 20μg/ml etoposide for 24h, SW620 cells transfected with USP9X- shRNA showed an obvious higher apoptosis rate than the control cells. Conclusion: RNA interference of USP9X can enhance the sensitive of human colon cancer cell line SW620 to etoposide. The apoptosis of SW620 that induced by etoposide was severely increased. USP9X gene may become the new target of gene therapy for colorectal cancer.
Objctives: To investigate the effect of USP9X on apoptosis of colon cancer induced by etoposide and its potential mechanism. Methods: USP9X small hairpin RNA(shRNA)sequence and control shRNA sequence were designed and synthesized, and the interference effective of USP9X- shRNA was detected by semi- quantitative RT- PCR and Western blotting. After transfected with USP9X- shRNA and shcontrol, SW620 cells were treated with etoposide, the de-tection of c- PARP were used to analysis the apoptosis of the cell. Results: Semi- quantitative RT- PCR and Western blot showed that the mRNA and protein level of USP9X were downregulated in SW620 cells transfected with USP9X- shRNA, and the inhibition rate reach to 70%. The shRNA sequence worked well for the following experiments. After treated with 20μg/ml etoposide for 24h, SW620 cells transfected with USP9X- shRNA showed an obvious higher apoptosis rate than the control cells. Conclusion: RNA interference of USP9X can enhance the sensitive of human colon cancer cell line SW620 to etoposide. The apoptosis of SW620 that induced by etoposide was severely increased. USP9X gene may become the new target of gene therapy for colorectal cancer.
引文
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[2]Hanahan D,Weinberg RA.Hallmarks of cancer:the next generation[J].Cell,2011,144(5):646-674.
[3]Vucic D,Dixit VM,Wertz IE.Ubiquitylation in apoptosis:a posttranslational modification at the edge of life and death[J].Nat Rev Mol Cell Biol,2011,12(7):439-452.
[4]Schwickart M,Huang X,Lill JR,et al.Deubiquitinase USP9X stabilizes MCL1 and promotes tumour cell survival[J].Nature,2010,463(7277):103-107.
[5]Luise C,Capra C,Donzelli M,et al.An atlas of altered expression of deubiquitinating enzymes in human cancer[J].PLoS One,2011,6(1):e15891.
[6]Dupont S,Mamidi A,Cordenonsi M,et al.FAM/USP9X,a deubiquitinating enzyme essential for TGFbeta signaling,controls Smad4 monoubiquitination[J].Cell,2009,136(1):123-135.
[7]Zhang C,Cai TY,Zhu H,et al.Synergistic antitumor activity of gemcitabine and ABT-737 in vitro and in vivo through disrupting the interaction of USP9X and Mcl-1[J].Mol Cancer Ther,2011,10(7):1264-1275.
[8]Gomez-Bougie P,Menoret E,Juin P,et al.Noxa controls Muledependent Mcl-1 ubiquitination through the regulation of the Mcl-1/USP9X interaction[J].Biochem Biophys Res Commun,2011,413(3):460-464.
[9]Nagai H,Noguchi T,Homma K,et al.Ubiquitin-like sequence in ASK1 plays critical roles in the recognition and stabilization by USP9X and oxidative stress-induced cell death[J].Mol Cell,2009,36(5):805-818.
[10]Grou CP,Francisco T,Rodrigues TA,et al.Identification of ubiquitin-specific protease 9X(USP9X)as a deubiquitinase acting on the ubiquitin-peroxin 5(PEX5)thioester conjugate[J].J Biol Chem,2012,287(16):12815-12827.
[11]Rott R,Szargel R,Haskin J,et al.alpha-Synuclein fate is determined by USP9X-regulated monoubiquitination[J].Proc Natl Acad Sci USA,2011,108(46):18666-18671.
[12]张娇,陈爱平,戚玉言,等.靶向EGFR的干扰RNA对卵巢癌耐药细胞凋亡的影响[J].中国肿瘤生物治疗杂志,2009,16(6):619-623.
[13]褚波,黄雪峰,唐云明.慢病毒载体及其应用进展[J].生物医学工程学杂志,2008,25(1):224-226.