海洋抗腫瘤多肽CS5931靶向作用于Enolase1抑制結(jié)腸癌生長與轉(zhuǎn)移的分子機制研究
發(fā)布時間：2018-07-20 19:21
【摘要】：CS5931是我們課題組從薩氏海鞘(Ciona savignyi)中分離得到的抗腫瘤多肽,分子量為5931Da。前期研究表明,CS5931對多種腫瘤細胞的生長具有顯著抑制活性。進一步研究證實CS5931可通過線粒體途徑誘導(dǎo)腫瘤細胞凋亡;可以抑制VEGF(Endothelial Growth Factor)及金屬蛋白酶類(Matrix Metalloproteinases,MMPs)的表達,但是其作用靶點尚不清楚。本研究利用細胞免疫熒光技術(shù)研究了CS5931在人結(jié)腸癌HCT116細胞的分布,證實CS5931主要作用在細胞膜上;利用SDS-PAGE、Pull-down、LC-MS/MS技術(shù)研究了CS5931作用的靶點,SDS-PAGE結(jié)果顯示與CS5931結(jié)合的靶蛋白分子量約50kDa,LC-MS/MS表明靶蛋白與人烯醇化酶1(Enolase 1,ENO1)具有高度的相似性。Western blotting證實靶蛋白可以與ENO1單克隆抗體特異性結(jié)合。利用表面等離子共振實驗(Surface plasmin resonance,SPR)進一步證實,CS5931可以與ENO1相互作用,二者的親和常數(shù)KD值為36.32μM。烯醇化酶是糖類代謝中的關(guān)鍵酶之一,能催化2-磷酸甘油酸脫水生成為磷酸烯醇式丙酮酸,存在三種亞型:α-烯醇化酶(Enolase 1,ENO1),β-烯醇化酶(Enolase 3,ENO3),γ-烯醇化酶(Enolase 2,ENO2)。已有報道表明,ENO1能促進腫瘤細胞的遷移和侵襲,因此我們采用Transwell實驗證實研究了CS5931對腫瘤細胞遷移與侵襲的影響,結(jié)果表明,CS5931可以顯著抑制結(jié)腸癌細胞HCT116細胞的遷移和侵襲。為了檢測CS5931抑制人結(jié)腸癌HCT116細胞的遷移和侵襲與ENO1的相關(guān)性,我們研究了過表達ENO1后CS5931對腫瘤細胞遷移與侵襲的影響,結(jié)果表明,過表達ENO1可以顯著增加腫瘤細胞的遷移與侵襲,而ENO1過表達可以逆轉(zhuǎn)CS5931對腫瘤細胞遷移與侵襲的影響。ENO1能分別與纖維蛋白溶酶原(plasminogen,PLG)、尿激酶纖維蛋白溶酶原激活劑(urokinase-type plasminogen activator,uPA)和尿激酶纖維蛋白溶酶原激活劑受體(urokinase-type plasminogen activator receptor,uPAR)結(jié)合,此外,PA/PLG系統(tǒng)是調(diào)節(jié)細胞外基質(zhì)降解的關(guān)鍵。我們用點雜交實驗研究了CS5931對ENO1與細胞因子結(jié)合的影響,結(jié)果顯示,點雜交實驗表明CS5931有效抑制ENO1與PLG,uPA/uPAR的結(jié)合。以上實驗說明,CS5931可以與ENO1特異性結(jié)合,二者的相互作用影響腫瘤細胞遷移和侵襲。綜上所述,我們的研究結(jié)果證實,CS5931可以與ENO1發(fā)生特異性相互作用,并通過影響ENO1的功能,抑制腫瘤細胞的遷移與侵襲。這說明CS5931與ENO1的相互作用是影響腫瘤細胞功能的重要因素。本研究對開發(fā)多肽類抗腫瘤藥物奠定了一定基礎(chǔ),對全面認識ENO1的生物學(xué)功能具有重要價值,對發(fā)展ENO1作為抗腫瘤藥物靶標也具有重要意義。
[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: 偽 -enolase (Enolase 1), 尾 -enolase (Enolase 3), and 緯 -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.
【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R96
本文編號：2134512
[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: 偽 -enolase (Enolase 1), 尾 -enolase (Enolase 3), and 緯 -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.
【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R96
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