siRNA介导的NFBD1表达沉默对HeLa细胞增殖与凋亡的影响  

Effects of siRNA-mediated NFBD1 Silencing on Cellular Growth and Apoptosis in HeLa Cells

作　　者：卜友泉[1] 杨正梅[2] 兰欢[1] 镇磊[1] 刘革力[1] 宋方洲[1] 

机构地区：[1]重庆医科大学生物化学与分子生物学教研室,重庆400016 [2]重庆医科大学实验动物中心,重庆400016

出　　处：《中国生物化学与分子生物学报》2008年第10期943-949,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：重庆市科委自然科学基金(No.2007BB5302);重庆医科大学重点课题(No.XBZD200707);国家自然科学基金(No.30671008);国家公派留学基金(No.2004850010)资助~~

摘　　要：NFBD1,也称MDC1,是1个参与细胞内DNA损伤后细胞应答反应的重要分子.为探讨NFBD1在细胞增殖和凋亡中的作用及其作为分子靶点用于肿瘤治疗的潜在价值,本研究采用siRNA技术抑制NFBD1的表达,并观察了其对人宫颈癌细胞HeLa细胞增殖和细胞凋亡的影响.半定量RT-PCR和蛋白质印迹分析结果表明,筛选到的短链NFBD1 siRNA能有效抑制内源NFBD1的表达,抑制程度约100%.细胞生长曲线分析结果表明,siRNA介导的NFBD1表达沉默导致HeLa细胞生长增殖的显著抑制.FACS分析结果表明,NFBD1表达抑制导致sub-G1峰的出现,同时蛋白质印迹分析观察到了caspase 3和PARP(poly-ADP-ribose polymerase)的剪接激活,表明NFBD1表达抑制诱发了细胞凋亡.在该凋亡过程中,P53及其下游靶分子Bax和Puma的表达水平均没有发生明显变化,但Noxa的表达在mRNA和蛋白质水平上均显著上调,强烈提示该凋亡过程很可能是1个不依赖P53的凋亡途径,且Noxa的转录激活在该凋亡过程中可能起着重要作用.这些结果表明,NFBD1参与细胞生长增殖和凋亡的调节,是一个潜在的肿瘤治疗新靶点.NFBD1 （a nuclear factor with BRCT domain 1）, also defined as MDC1 （mediator of DNA damage checkpoint protein 1）, is a large nuclear protein that mainly participates in DNA damage. To investigate its role in cell proliferation and apoptosis, and its therapeutic potential against cancer, we used the specific siRNA duplexes to inhibit NFBD1 expression and determined the effects of NFBD1 depletion on cellular proliferation and apoptosis in HeLa cells. Semi-quantitative RT-PCR and immunoblotting analysis revealed that NFBD1 expression was efficiently inhibited by specific siRNA against NFBD1 in HeLa cells. The siRNA- mediated NFBD 1 depiction caused a significant inhibition of cell proliferation. Furthermore, the induction of apoptosis was also observed in NFBD1-depleted HeLa cells, as indicated by the sub-G1 population and cleavage of both easpase 3 and PARP（poly-ADP-ribose polymerase）. Additionally, the expression levels of p53 as well as its downstream targets Bax and Puma remained unchanged, whereas the expression levels of puma was significantly upregulated in NFBD1-depleted cells, suggesting that the NFBD1 depletion-induced apoptosis is independent of P53 and probably mediated by upregulation of Noxa. Taken together, these results indicated that, beside its well-known role in DNA damage, NFBD1 is also involved in the regulation of cell proliferation and apoptosis, and might serve as a therapeutic target in cancer treatment.

关 键 词：NFBD1 SIRNA 细胞增殖 细胞凋亡 

分 类 号：Q71[生物学—分子生物学] R73[医药卫生—肿瘤]