Rasfonin潜在结合蛋白的发现及其对胰腺癌细胞增殖影响的初步研究

Preliminary study on the discovery of potential binding proteins of rasfonin and its effect on the proliferation of pancreatic cancer cells

目的寻找对Kras突变胰腺癌增殖具有选择性抑制作用的化合物rasfonin的结合蛋白,研究其潜在结合蛋白对胰腺癌Panc-1细胞增殖的影响,为其抗胰腺癌的机制研究提供线索。方法运用依赖于靶点稳定性的药物亲和反应(DARTS)方法获得可能与rasfonin结合的耐酶解电泳条带,经质谱鉴定、Mascot分析后得到潜在结合蛋白。设计针对这些蛋白的siRNA片段,qPCR法检测siRNA干扰效率。选择干扰效率最高的siRNA转染Panc-1细胞,CCK8法检测细胞增殖率。结果对得到的电泳条带进行解析后得到11种与肿瘤密切相关的蛋白,对相关基因进行siRNA干扰实验后检测细胞增殖情况,结果显示敲降YBX1、LAP3基因抑制Panc-1细胞增殖;敲降PPP2R5E基因促进Panc-1细胞增殖;敲降其他基因对Panc-1细胞增殖无明显影响。结论 YBX1、LAP3和PPP2R5E基因对Panc-1细胞的增殖具有明显影响,但其与rasfonin发挥抗肿瘤作用的关系尚需深入研究。

Objective To find out binding proteins of rasfonin,a compound that specifically inhibits the proliferation of Kras mutant pancreatic cancer cells,and study the effect of the potential binding proteins on the proliferation of pancreatic cancer cells to give clues to further exploration of the anti-pancreatic cancer mechanism of rasfonin. Methods The drug affinity responsive target stability(DARTS)method was used to obtain an enzymatically resistant electrophoresis band that may bind to rasfonin. After mass spectrometry identification and Mascot analysis,potential binding proteins were obtained. The siRNA fragments targeting these proteins were designed and qPCR was used to detect siRNA interference efficiency. Panc-1 cells were transfected with the siRNA with the highest interference efficiency,and the cell proliferation rate was detected by CCK8 method. Results The 11 proteins closely related to tumor were obtained after analysis of the obtained electrophoresis band. The proliferation of the Panc-1 cells was detected after siRNA interference assay. The results showed that knockdown of YBX1 and LAP3 genes inhibited the proliferation of Panc-1 cells;knockdown of PPP2 R5 E gene promoted the proliferation of Panc-1 cells;knockdown of other genes had no significant effects on the proliferation of Panc-1 cells. Conclusion YBX1,LAP3 and PPP2 R5 E genes showed significant effects on the proliferation of Panc-1 cells,but the relation between these genes and the anti-tumor effect of rasfonin need to be further investigated.