Granulin A与ENO1的相互作用及其抑制肿瘤生长和转移的分子机制研究

Peptides play a crucial role in many physiological processes. However, a small number of peptides have been commercialized as drugs because of their limited in low yield and poor bioavailability. It is promising to develop human peptides as drugs due to its low immunogenicity as well as its safety. In our previous study, we have confirmed that granulin A (GRN A) displays potent cytotoxicity to several human cancer cells with nano molar level. Treatment of cancer cells results in inhibition of invasion and migration of cancer cells. GRN A is able to interact with enolase1 (ENO1) specifically as determined by pull-down, MS and Western blotting analysis. However, little is known about the property of the interaction. It is also unclear that if the interaction between GRN A and ENO1 affects the function of cancer cells. In this project, we plan to: 1) elucidate the property of GRN A and ENO1 using molecular docking approach, CD spectra analysis as well as site-directed mutation; 3)dissect the function of cancer cells affected by the interaction; 2) study the effect of GRN A on the aerobic glycolysis in cancer cells; 4) investigate the interaction of GRN A and ENO1 in vivo. The study is important for the understanding the molecular mechanism of ENO1. This study also provides a novel strategy for developing peptides from human source as novel anticancer agents.

人体来源的抗肿瘤多肽由于没有免疫原性、副作用小，发现具有抗肿瘤作用的人源多肽具有重要意义。前期研究发现，人颗粒体蛋白A（GRN A）对多种肿瘤细胞具有显著的抑制作用，并能抑制肿瘤细胞的迁移与侵袭，但其作用的靶点尚不清楚。我们采用pull-down/MS以及Western blotting分析，证实GRN A可以与烯醇化酶1(ENO1）发生特异性结合。但二者相互作用的结构特征及其对肿瘤细胞生物功能的影响尚不清楚。本研究拟采用分子对接、定点突变、荧光色谱等技术阐明GRN A对ENO1结构的影响；研究GRN A与ENO1相互作用对肿瘤细胞能量代谢的影响； 研究在ENO1敲除及过表达条件下GRN A对肿瘤细胞增殖、迁移及侵袭的变化规律；采用裸鼠移植性模型揭示GRN A与ENO1的体内作用规律。研究结果对发展ENO1作为抗肿瘤药物靶标具有重要意义，对GRN A作为新型抗肿瘤药物的研发也具有重要价值。

Granulin A是颗粒体蛋白包含的多肽片段，前期研究证实Granulin A可以与烯醇式酶1 （enolase1, ENO1）发挥相互作用，诱导肿瘤细胞自噬，但其具体的分子机制上不清楚。本研究利用SPR, Western blot及LC-MS等技术对二者的相互作用进行了进一步分析，发现二者可以特异性结合，为了研究二者的结合对肿瘤细胞生长作用的影响，我们采用Transwell 实验测定了Granulin A 对肿瘤细胞迁移与管腔形成的影响，发现Granulin A可以显著抑制肿瘤细胞的迁移与管腔形成，同时Granulin A还可以抑制糖酵解的的关键酶的表达。 构建了ENO1高表达肿瘤细胞，研究了在ENO1过表达条件下 Granulin A的功能，发现ENO1过表达可以逆转Granulin A对肿瘤细胞迁移与管腔形成的抑制作用，点杂技实验证实granulin A可以抑制ENO1与uPA, uPAR以及OVA等转移相关因子的相互作用。同时本研究还证实Granulin A与顺铂联合应用具有显著协同作用，在Granulin A存在条件下，顺铂对胰腺癌细胞的敏感性显著提高，过表达ENO1可以部分逆转二者的协同作用。本研究为发展Granulin A作为抗肿瘤新药提供了依据，同时证实ENO1可以作为抗肿瘤药物的靶点。课题共发表SCI论文4篇，申请国家发明专利一项。