海洋抗肿瘤多肽CS5931靶向作用于Enolase1抑制结肠癌生长与转移的分子机制研究

发布时间：2018-07-20 19:21

【摘要】：CS5931是我们课题组从萨氏海鞘(Ciona savignyi)中分离得到的抗肿瘤多肽,分子量为5931Da。前期研究表明,CS5931对多种肿瘤细胞的生长具有显著抑制活性。进一步研究证实CS5931可通过线粒体途径诱导肿瘤细胞凋亡;可以抑制VEGF(Endothelial Growth Factor)及金属蛋白酶类(Matrix Metalloproteinases,MMPs)的表达,但是其作用靶点尚不清楚。本研究利用细胞免疫荧光技术研究了CS5931在人结肠癌HCT116细胞的分布,证实CS5931主要作用在细胞膜上;利用SDS-PAGE、Pull-down、LC-MS/MS技术研究了CS5931作用的靶点,SDS-PAGE结果显示与CS5931结合的靶蛋白分子量约50kDa,LC-MS/MS表明靶蛋白与人烯醇化酶1(Enolase 1,ENO1)具有高度的相似性。Western blotting证实靶蛋白可以与ENO1单克隆抗体特异性结合。利用表面等离子共振实验(Surface plasmin resonance,SPR)进一步证实,CS5931可以与ENO1相互作用,二者的亲和常数KD值为36.32μM。烯醇化酶是糖类代谢中的关键酶之一,能催化2-磷酸甘油酸脱水生成为磷酸烯醇式丙酮酸,存在三种亚型:α-烯醇化酶(Enolase 1,ENO1),β-烯醇化酶(Enolase 3,ENO3),γ-烯醇化酶(Enolase 2,ENO2)。已有报道表明,ENO1能促进肿瘤细胞的迁移和侵袭,因此我们采用Transwell实验证实研究了CS5931对肿瘤细胞迁移与侵袭的影响,结果表明,CS5931可以显著抑制结肠癌细胞HCT116细胞的迁移和侵袭。为了检测CS5931抑制人结肠癌HCT116细胞的迁移和侵袭与ENO1的相关性,我们研究了过表达ENO1后CS5931对肿瘤细胞迁移与侵袭的影响,结果表明,过表达ENO1可以显著增加肿瘤细胞的迁移与侵袭,而ENO1过表达可以逆转CS5931对肿瘤细胞迁移与侵袭的影响。ENO1能分别与纤维蛋白溶酶原(plasminogen,PLG)、尿激酶纤维蛋白溶酶原激活剂(urokinase-type plasminogen activator,uPA)和尿激酶纤维蛋白溶酶原激活剂受体(urokinase-type plasminogen activator receptor,uPAR)结合,此外,PA/PLG系统是调节细胞外基质降解的关键。我们用点杂交实验研究了CS5931对ENO1与细胞因子结合的影响,结果显示,点杂交实验表明CS5931有效抑制ENO1与PLG,uPA/uPAR的结合。以上实验说明,CS5931可以与ENO1特异性结合,二者的相互作用影响肿瘤细胞迁移和侵袭。综上所述,我们的研究结果证实,CS5931可以与ENO1发生特异性相互作用,并通过影响ENO1的功能,抑制肿瘤细胞的迁移与侵袭。这说明CS5931与ENO1的相互作用是影响肿瘤细胞功能的重要因素。本研究对开发多肽类抗肿瘤药物奠定了一定基础,对全面认识ENO1的生物学功能具有重要价值,对发展ENO1作为抗肿瘤药物靶标也具有重要意义。
[Abstract]:CS5931 is an antitumor polypeptide isolated from Ciona savignyi. The molecular weight of CS5931 is 5931 Daa. Previous studies have shown that CS5931 has a significant inhibitory activity on the growth of various tumor cells. Further studies have confirmed that CS5931 can induce apoptosis of tumor cells through mitochondrial pathway, and inhibit the expression of VEGF (Endothelial growth Factor) and Matrix Metalloproteinases (MMPs), but the target of CS5931 is not clear. In this study, the distribution of CS5931 in human colon cancer HCT116 cells was studied by using cellular immunofluorescence technique, and it was proved that CS5931 mainly acted on the cell membrane. SDS-PAGEN Pull-downdown LC-MS / MS technique was used to study the target of CS5931. The results of SDS-PAGE showed that the target protein bound to CS5931 had a molecular weight of about 50 kDaLC-MS-MS, which indicated that the target protein had a high similarity with human enolase 1 (Enolase 1 / ENO1). Western blotting confirmed that the target protein could specifically bind to ENO1 monoclonal antibody. Surface plasmin resonance spectroscopy (SPR) showed that CS5931 could interact with ENO1, and their affinity constant KD was 36.32 渭 M. Enolase is one of the key enzymes in carbohydrate metabolism. It can catalyze the dehydration of 2-phosphoglyceric acid to phosphoenolpyruvate. There are three subtypes: 伪 -enolase (Enolase 1), 尾 -enolase (Enolase 3), and 纬 -enolase (Enolase 2 (ENO2). It has been reported that ENO1 can promote the migration and invasion of tumor cells. Therefore, we have studied the effect of CS5931 on the migration and invasion of tumor cells by Transwell experiment. The results show that CS5931 can significantly inhibit the migration and invasion of human colon cancer cell line HCT116. In order to investigate the relationship between the inhibition of migration and invasion of human colon cancer HCT116 cells by CS5931 and ENO1, we studied the effect of CS5931 on the migration and invasion of human colon cancer HCT116 cells after overexpression of ENO1. Overexpression of ENO1 could significantly increase the migration and invasion of tumor cells. ENO1 overexpression could reverse the effect of CS5931 on tumor cell migration and invasion. ENO1 could bind to plasminogen (PLG), urokinase plasminogen activator (urokinase-type plasminogen) and urokinase plasminogen activator receptor (urokinase-type plasminogen activator receptor upar), respectively. In addition, the PA-P / PLG system is the key to regulate the degradation of extracellular matrix. The effect of CS5931 on the binding of ENO1 to cytokines was studied by dot hybridization. The results showed that CS5931 could effectively inhibit the binding of ENO1 to PLGUPA / upar. These results suggest that CS5931 can specifically bind to ENO1, and the interaction between them affects the migration and invasion of tumor cells. In conclusion, our results show that CS5931 can specifically interact with ENO1 and inhibit the migration and invasion of tumor cells by affecting the function of ENO1. This suggests that the interaction between CS5931 and ENO1 is an important factor affecting the function of tumor cells. This study has laid a foundation for the development of polypeptide antineoplastic drugs and has important value in understanding the biological function of ENO1 and developing ENO1 as a target of anti-tumor drugs.
【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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