siRNA介导的NFBD1表达沉默对HeLa细胞增殖与凋亡的影响

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，P-53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．

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

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 NFBD1 depletion 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 caspase 3 and PARP（poly-ADP-ribose polymerase）. Additionally, the expression levels of p-53 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.