核因子蛋白90敲减对肝癌细胞增殖影响及机制的研究

Effects of nuclear factor 90 knockdown on proliferation of hepatocellular carcinoma cells and its mechanism

目的探究核因子蛋白90(NF90)敲减对肝癌细胞增殖影响并探讨其可能机制。方法将靶向NF90的寡核苷酸链克隆至PLKO质粒后,包装出靶向敲减NF90的慢病毒shRNA(带有绿色荧光标签及G418抗性)。将肝癌细胞QGY-7703、SMMC-7721分为对照组及干扰组,分别感染包含随机序列shRNA和靶向NF90 shRNA的病毒液。48 h后流式分选出带有绿色荧光的阳性细胞,G418筛选阳性单克隆细胞株,Western blotting鉴定NF90的蛋白表达水平,最终在对照组中选取一株细胞命名shRNA-ns,在干扰组中选取NF90敲减效果良好且稳定的两株细胞命名为shRNA-1、shRNA-2。采用CCK-8法检测各株细胞的增殖情况,克隆形成试验分析各株细胞的克隆形成能力。质谱分析预测NF90的相关结合蛋白,内、外源免疫共沉淀确定NF90与多聚ADP核糖合成酶(PARP1)的关系,荧光定位分析NF90与PARP1在细胞内的定位情况。结果 shRNA-1、shRNA-2细胞中的NF90表达水平显著低于shRNA-ns细胞,表明包装的慢病毒敲减NF90效果良好。与shRNA-ns细胞比较,shRNA-1、shRNA-2细胞的生长缓慢,克隆形成数减少,差异均有统计学意义(P<0.01)。NF90与PARP1在细胞内相互结合并且共定位于细胞核。结论敲减NF90可抑制肝癌细胞增殖,其机制可能与PARP1相互作用有关。

Objective To investigate the effect of nuclear factor 90 （NF90） knockdown on proliferation of hepatocellular carcinoma （HCC） cell and explore its mechanism.Methods The oligonucleotides targeting NF90 were cloned into the PLKO plasmid and packaged with lentiviral shRNA to knockdown NF90 （with green fluorescent labels and G418 resistance）.The hepatocellular carcinoma cells QGY-7703 and SMMC-7721 were divided into control group and intervention group.The virus containing random sequence shRNA and targeting NF90 shRNA were infected, respectively.After 48 h, positive cells were stained with green fluorescence, and the positive monoclonal cell lines were screened by G418.The expression level of NF90 protein was identified by Western blotting.Finally, a cell line named shRNA-ns was selected in the control group.Two cell lines of NF90 knockdown named shRNA-1 and shRNA-2.Cell growth activity was detected by CCK-8 and clone formation analysis.The relationship between NF90 and PARP1 was detected by fluorescence localization assay.Results The expression of NF90 in shRNA-1 and shRNA-2 cells was significantly lower than that in shRNA-ns cells, indicating that the knockdown of NF90 by lentivirus was effective.Compared with shRNA-ns cells, shRNA-1 and shRNA-2 cells were slow to grow and the number of clones was decreased with statistically significant difference （P〈0.01）.NF90 and PARP1 bind to each other in the cell and co-localize in the nucleus.Conclusion NF90 knockdown inhibits HCC cell proliferation, and NF90 may affect HCC cell proliferation by interacting with PARP1.