颗粒体蛋白A靶向作用于烯醇化酶1抑制肝癌细胞侵袭和迁移的分子机制研究

发布时间：2018-04-18 06:21

本文选题：肝癌 + 颗粒体蛋白A　；参考：《首都医科大学》2017年博士论文

【摘要】：肝细胞癌(Hepatocellular carcinoma,HCC)是世界上常见的恶性肿瘤之一,因其发病率高、复发转移率高、死亡率高等特点,严重威胁着人类的生命健康。我国是世界上肝癌第一大国,其男性和女性发病率分别居第3、第5位,死亡率分别居第2、第4位。目前,外科手术依然是肝癌患者获得长期生存的主要治疗方法,但由于术后较高的复发和转移率,其疗效并不理想。因此,对肝癌转移复发机制进行深入研究,探索治疗干预的新方法对增加肝癌患者的生存率具有十分重要的意义。多肽在许多生理、生化过程中具有十分重要的作用。然而受限于多肽类药物的含量和容易失活等特点,限制了这类药物的应用。人体来源的抗肿瘤多肽没有免疫原性,副作用小,因此发现此类多肽具有十分重要的意义。人颗粒体蛋白A(Granulin A,GRN A)是由颗粒体蛋白前体(Progranulin,PGRN)酶解产生的、分子量约为6 kDa的多肽。我们前期的研究发现,人GRN A对多种肿瘤细胞的生长具有显著的抑制作用,有希望成为一种新型的抗肿瘤药物,但GRN A作用的机制尚不清楚,阐明GRN A的作用靶点对发展人源化多肽作为抗肿瘤药物具有重要价值。为了鉴定GRN A的作用靶点,我们采用激光共聚焦技术研究了GRN A在HepG-2细胞的定位,发现GRN A在胞膜和胞质上均有分布;采用SDS-PAGE、Pull-down、LC-MS/MS等技术鉴定了与GRN A相互作用的靶蛋白。SDS-PAGE分析表明,与GRN A相互结合的靶蛋白分子量约50 kDa,LC-MS/MS证实靶蛋白与烯醇化酶1(Enolase 1,ENO1)有高度的相似性;进而我们采用Western Blotting分析,证实靶蛋白可以与ENO1单克隆抗体特异性结合;采用表面等离子共振技术(Surface plasmon resonance,SPR)分析两者之间的相互作用,证实GRN A与ENO1之间的亲和常数(Km)为56.04μM。这些结果有力地证明:grna可以与eno1发生特异性相互作用。eno1是糖类代谢中的关键酶之一,可以催化2-磷酸-d-甘油酸(2-phosphate-d-glycerate,2-pg)转化为磷酸烯醇式丙酮酸(phosphoricacid-pyruvate,pep)。为了研究grna和eno1相互作用的生物学功能,我们首先研究了grna对糖代谢的影响。此前的报道表明,enoblock(ap-iii-a4)可以与eno1结合并抑制eno1的功能。因此我们比较了grna与enoblock对葡萄糖摄取的影响。结果表明grna处理后的hepg-2细胞糖摄取量明显升高,用浓度为2.5、5.0、7.5、10.0μm的grna处理hepg-2细胞48h后,葡萄糖摄取量分别从0.94±0.43增加到1.89±0.46、2.01±0.23、3.00±0.26、3.10±0.27mg/mgprotein。此外,葡萄糖摄取量随grna作用时间的延长而增加,用5μmgrna处理hepg-2细胞6、12、24、48、72h后,摄糖量分别为0.20±0.12、0.58±0.12、1.36±0.08、2.10±0.05、3.68±0.16mg/mgprotein。westernblotting证实grna是通过抑制糖异生关键酶磷酸烯醇式丙酮酸羧激酶1(phosphoenolpyruvatecarboxykinase1,pck1)和磷酸烯醇式丙酮酸羧激酶2(phosphoenolpyruvatecarboxykinase2,pck2)的表达来实现葡萄糖摄取量增加的,这种作用与enoblock的作用一致,说明grna也起到eno1抑制剂的功能。已有的报道表明,eno1在多种肿瘤细胞高表达,与肿瘤转移具有关系密切。为了研究二者之间的相互作用是否影响肿瘤细胞的侵袭和转移,我们采用细胞划痕及transwell实验,研究了grna对肝癌hepg-2细胞的影响。细胞划痕实验结果证实grna处理可以显著抑制hepg-2细胞的迁移作用,用2μmgrna处理hepg-2细胞12、24h,肿瘤细胞的迁移率分别为29.60±2.33%和40.97±1.23%,显著低于对照组。transwell实验进一步证实grna对hepg-2细胞迁移具有显著的抑制作用,用0、0.5、1.0、2.0μmgrna分别处理hepg-2细胞48h后,迁移到transwell小室下层的数目由378±28分别减少到297±35、210±13、146±17。enoblock处理也呈现类似的结果。另外,grna可以显著抑制hepg-2细胞的侵袭作用,且呈剂量依赖性。0、0.5、1.0、2.0μm浓度的grna处理hepg-2细胞后,小室下层的数目由208±16减少到167±16、115±3、62±9,这种作用与enoblock处理后对hepg-2细胞侵袭的抑制作用类似。上述结果说明,grna与eno1抑制剂enablock的作用一样,可以抑制肿瘤细胞的迁移与侵袭。为了研究grna对肿瘤细胞功能的影响与eno1的相关性,我们研究了在eno1过表达条件下grna对肝癌细胞hepg2功能的影响。发现高表达eno1可以拮抗grna对hepg-2细胞迁移和侵袭的抑制作用。transwell实验结果显示,过表达eno1可以增加肝癌hepg-2细胞的迁移;迁移细胞的数目从227±15增加到427±49,这说明eno1有促进细胞迁移的能力。grna处理肝癌细胞后,细胞的迁移数由227±15降低至144±21,但在eno1高表达的肝癌细胞中,grna处理后,细胞迁移数从144±21增加到346±46。同样,过表达eno1可以拮抗grna对hepg-2细胞侵袭的抑制作用,在eno1高表达的肝癌细胞中,hepg-2侵袭数目从67±10增加到112±16。以上实验结果表明grna可以与eno1结合,并通过影响eno1的功能抑制肿瘤细胞的迁移和侵袭。为了研究grna对凋亡和糖异生相关蛋白表达的影响和eno1的相关性,我们研究了eno1过表条件下grna对凋亡和糖异生相关蛋白表达的影响。我们发现,grna处理过后的细胞抗凋亡蛋白akt、bcl-xl、c-myc蛋白明显减少;过表达eno1后蛋白浓度明显增加。而相同浓度grna处理hepg-2细胞后,过表达eno1的细胞中的抗凋亡蛋白浓度明显高于非过表达eno1细胞,这说明grna是通过与eno1结合作用诱导细胞凋亡。相似地,grna可以通过作用eno1调控pck1、pck2影响hepg-2细胞葡萄糖摄取。以上实验结果证明grna可以与eno1结合,并通过影响eno1的功能影响相关抗凋亡蛋白和糖异生蛋白的表达。综上所述,我们的研究结果证实,grna可以诱导肿瘤细胞凋亡、抑制肿瘤细胞的迁移与侵袭,增加肿瘤细胞葡萄糖的摄取。grna可以与eno1发生特异性相互作用,eno1过表达可以促进肿瘤细胞的迁移与侵袭,并能拮抗grna对肿瘤细胞凋亡、迁移与侵袭及葡萄糖摄取的功能的影响。这说明grna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]:Hepatocellular carcinoma (Hepatocellular, carcinoma, HCC) is one of the most common malignancies in the world, because of its high incidence, high recurrence rate, high mortality rate, a serious threat to human life and health. China is the world's first big liver cancer, the incidence of male and female respectively in third, Fifth. The mortality rate ranked second, fourth. At present, surgery is still the main method for the treatment of hepatocellular carcinoma patients with long-term survival, but due to the high rate of recurrence and metastasis after operation, the curative effect is not ideal. Therefore, the mechanism of HCC metastasis. Further research, to explore a new method of treatment is of great significance to to increase the survival rate of patients with HCC. The polypeptide in many physiological and biochemical processes plays an important role. However, due to the content of polypeptide drugs and easy inactivation etc., restrict the application of these drugs. The anti tumor peptide from no immunogenicity, little side effect, therefore has very important significance. This kind of polypeptide A protein particles (Granulin A, GRN A) is a granular protein precursor (Progranulin, PGRN) enzymatic peptide, molecular weight of about 6 kDa. Our previous study found that significantly inhibited the growth of the GRN A on a variety of tumor cells, there is hope to become a new anticancer drug, but the mechanism of GRN A function is not clear, the target GRN A to clarify anti-tumor drugs has important value for the development of humanized polypeptides as to. Target identification of GRN A, we used confocal laser positioning technology research of GRN A in HepG-2 cells, GRN A were distributed in the cytoplasm; using SDS-PAGE, Pull-down, LC-MS/MS and other technical appraisal by the target protein.SDS-PAGE interaction with GRN A Analysis shows that the molecular target protein combined with GRN A about 50 kDa, LC-MS/MS confirmed that the target protein with enolase 1 (Enolase 1, ENO1) have a high degree of similarity; then we use Western Blotting analysis confirmed that the target protein could bind with ENO1 monoclonal antibody; using surface plasmon resonance technology (Surface plasmon resonance, SPR) analysis of the interaction between the two, the affinity constant between GRN and A confirmed that ENO1 (Km) 56.04 M. these results strongly suggest that gRNA can specific interaction between.Eno1 and eno1 is one of the key enzymes in the metabolism of carbohydrate, can catalyze the phosphorylation of 2- (2-phosphate-d-glycerate, -d- glycerol acid 2-PG) converted to phosphoenolpyruvate (phosphoricacid-pyruvate, PEP). In order to study the biological function of gRNA and eno1 interaction, we first studied the effect of gRNA on glucose metabolism. Previous reports Show that enoblock (ap-iii-a4) can bind to eno1 and inhibit the function of eno1. We compared the effects of gRNA and enoblock on glucose uptake. The results showed that HepG-2 cell glucose intake after treatment with gRNA significantly increased with concentration of 2.5,5.0,7.5,10.0 m gRNA in HepG-2 cells treated with 48h after glucose intake respectively from 0.94. 0.43 to 1.89 + 0.46,2.01 + 0.23,3.00 + 0.26,3.10 + 0.27mg/mgprotein. in addition, glucose uptake increased with the prolongation of gRNA time, with 5 mgrna treatment of HepG-2 cells after 6,12,24,48,72h, sugar intake were 0.20 + 0.12,0.58 + 0.12,1.36 + 0.08,2.10 + 0.05,3.68 + 0.16mg/mgprotein.westernblotting gRNA was confirmed by inhibition of gluconeogenesis enzyme phosphoenolpyruvate pyruvate carboxylase kinase 1 (phosphoenolpyruvatecarboxykinase1, PCK1) and phosphoenolpyruvate carboxykinase (phosphoenolpyruvatecar 2 Boxykinase2, pck2) expression of glucose uptake, consistent with a role this interaction with enoblock, indicating that gRNA plays eno1 inhibitor function. Previous reports showed that high expression of eno1 in tumor cells, and tumor metastasis has a close relationship. In order to study the interaction between the two effects of tumor cells the invasion and metastasis, we use Transwell and cell scratch experiments, the effects of gRNA on hepatocellular carcinoma HepG-2 cells. Cell scratch experimental results confirmed the transfer of gRNA treatment can significantly inhibit HepG-2 cells, HepG-2 cells were treated with 2 mgrna 12,24h, the migration rate of tumor cells was 29.60 + 2.33% and 40.97 + 1.23%, significantly compared with the control group.Transwell experiments further confirmed that gRNA migration has obvious inhibitory effect on HepG-2 cells, HepG-2 cells were treated respectively with 0,0.5,1.0,2.0 48h mgrna, moved The number of Transwell moved to the lower chamber by 378 + 28 + 35210 + 297 respectively reduced to 13146 + 17.enoblock treatment also showed similar results. In addition, gRNA can significantly inhibit the invasion of HepG-2 cells in a dose-dependent manner.0,0.5,1.0,2.0 M concentration of gRNA in HepG-2 cells after treatment, the number of the lower chamber by 208 + 16 reduced to 167 + 16115 + 3,62 + 9, inhibit the invasion of HepG-2 cells after enoblock treatment, and this effect is similar. These results indicated that gRNA and eno1 inhibitor enablock effect, can inhibit the migration and invasion of tumor cells. The correlation of gRNA effects on tumor cells and eno1, we study the effect of overexpression of gRNA under the condition of hepatocellular carcinoma cells in HepG2 eno1. Found that high expression of eno1 could antagonize the inhibitory effect of gRNA.Transwell experimental results on migration and invasion of HepG-2 cells. Showed that overexpression of eno1 could increase the migration of hepatocellular carcinoma HepG-2 cells; the number of migrating cells increased from 227 + 15 to 427 + 49, which shows that eno1 can promote the ability of.Grna cells treated with cell migration, migration cell number decreased from 227 + 15 to 144 + 21, but high expression in liver cancer eno1 in the cell, after gRNA treatment, the number of cell migration from 144 + 21 to 346 + 46., overexpression of eno1 could antagonize the inhibition of gRNA on invasion of HepG-2 cells, in hepatocellular carcinoma cells with high expression of eno1, HepG-2 invasion number from 67 + 10 to 112 + 16. increase above experimental results show that gRNA and eno1 can with, and influence the eno1 function of inhibiting tumor cell migration and invasion. In order to study the correlation expression of gRNA related protein on apoptosis and gluconeogenesis and eno1, we studied the effect of eno1 over expression of gRNA under the condition of expressed on apoptosis associated protein and sugar difference We found that the anti apoptotic protein Akt, Bcl-xL gRNA after treatment of cells, c-myc protein significantly decreased; expression of protein concentration increased significantly after eno1 and gRNA of the same concentration of HepG-2 cells after overexpression of anti apoptotic protein concentration in eno1 cells was significantly higher than that of non overexpression eno1 cells, indicating that gRNA is the role of through the combination of eno1 and induce cell apoptosis. Similarly, gRNA through the role of eno1 PCK1, pck2 influence the glucose uptake in HepG-2 cells. These results demonstrated that gRNA can bind to eno1, and through the influence of the functional effects of eno1 anti apoptosis protein and gluconeogenesis protein expression. In summary, our results confirm that gRNA can induction of tumor cell apoptosis, inhibit the migration and invasion of tumor cells, tumor cells increased glucose uptake in.Grna can specifically interacts with eno1, eno1 overexpression can promote The migration and invasion into tumor cells, and apoptosis of tumor cells against gRNA, effects of migration and invasion and glucose uptake function. This shows that the interaction between gRNA and eno1 is an important factor affecting tumor cell function. This study has important value for the comprehensive understanding of the biological function of ENO1, and also has an important significance for the development of ENO1 as anticancer drug targets.

【学位授予单位】：首都医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R96

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