本文選題：肝癌 + 顆粒體蛋白A��； 參考：《首都醫(yī)科大學(xué)》2017年博士論文

【摘要】：肝細(xì)胞癌(Hepatocellular carcinoma,HCC)是世界上常見的惡性腫瘤之一,因其發(fā)病率高、復(fù)發(fā)轉(zhuǎn)移率高、死亡率高等特點(diǎn),嚴(yán)重威脅著人類的生命健康。我國是世界上肝癌第一大國,其男性和女性發(fā)病率分別居第3、第5位,死亡率分別居第2、第4位。目前,外科手術(shù)依然是肝癌患者獲得長(zhǎng)期生存的主要治療方法,但由于術(shù)后較高的復(fù)發(fā)和轉(zhuǎn)移率,其療效并不理想。因此,對(duì)肝癌轉(zhuǎn)移復(fù)發(fā)機(jī)制進(jìn)行深入研究,探索治療干預(yù)的新方法對(duì)增加肝癌患者的生存率具有十分重要的意義。多肽在許多生理、生化過程中具有十分重要的作用。然而受限于多肽類藥物的含量和容易失活等特點(diǎn),限制了這類藥物的應(yīng)用。人體來源的抗腫瘤多肽沒有免疫原性,副作用小,因此發(fā)現(xiàn)此類多肽具有十分重要的意義。人顆粒體蛋白A(Granulin A,GRN A)是由顆粒體蛋白前體(Progranulin,PGRN)酶解產(chǎn)生的、分子量約為6 kDa的多肽。我們前期的研究發(fā)現(xiàn),人GRN A對(duì)多種腫瘤細(xì)胞的生長(zhǎng)具有顯著的抑制作用,有希望成為一種新型的抗腫瘤藥物,但GRN A作用的機(jī)制尚不清楚,闡明GRN A的作用靶點(diǎn)對(duì)發(fā)展人源化多肽作為抗腫瘤藥物具有重要價(jià)值。為了鑒定GRN A的作用靶點(diǎn),我們采用激光共聚焦技術(shù)研究了GRN A在HepG-2細(xì)胞的定位,發(fā)現(xiàn)GRN A在胞膜和胞質(zhì)上均有分布;采用SDS-PAGE、Pull-down、LC-MS/MS等技術(shù)鑒定了與GRN A相互作用的靶蛋白。SDS-PAGE分析表明,與GRN A相互結(jié)合的靶蛋白分子量約50 kDa,LC-MS/MS證實(shí)靶蛋白與烯醇化酶1(Enolase 1,ENO1)有高度的相似性;進(jìn)而我們采用Western Blotting分析,證實(shí)靶蛋白可以與ENO1單克隆抗體特異性結(jié)合;采用表面等離子共振技術(shù)(Surface plasmon resonance,SPR)分析兩者之間的相互作用,證實(shí)GRN A與ENO1之間的親和常數(shù)(Km)為56.04μM。這些結(jié)果有力地證明:grna可以與eno1發(fā)生特異性相互作用。eno1是糖類代謝中的關(guān)鍵酶之一,可以催化2-磷酸-d-甘油酸(2-phosphate-d-glycerate,2-pg)轉(zhuǎn)化為磷酸烯醇式丙酮酸(phosphoricacid-pyruvate,pep)。為了研究grna和eno1相互作用的生物學(xué)功能,我們首先研究了grna對(duì)糖代謝的影響。此前的報(bào)道表明,enoblock(ap-iii-a4)可以與eno1結(jié)合并抑制eno1的功能。因此我們比較了grna與enoblock對(duì)葡萄糖攝取的影響。結(jié)果表明grna處理后的hepg-2細(xì)胞糖攝取量明顯升高,用濃度為2.5、5.0、7.5、10.0μm的grna處理hepg-2細(xì)胞48h后,葡萄糖攝取量分別從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,葡萄糖攝取量隨grna作用時(shí)間的延長(zhǎng)而增加,用5μmgrna處理hepg-2細(xì)胞6、12、24、48、72h后,攝糖量分別為0.20±0.12、0.58±0.12、1.36±0.08、2.10±0.05、3.68±0.16mg/mgprotein。westernblotting證實(shí)grna是通過抑制糖異生關(guān)鍵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)的表達(dá)來實(shí)現(xiàn)葡萄糖攝取量增加的,這種作用與enoblock的作用一致,說明grna也起到eno1抑制劑的功能。已有的報(bào)道表明,eno1在多種腫瘤細(xì)胞高表達(dá),與腫瘤轉(zhuǎn)移具有關(guān)系密切。為了研究二者之間的相互作用是否影響腫瘤細(xì)胞的侵襲和轉(zhuǎn)移,我們采用細(xì)胞劃痕及transwell實(shí)驗(yàn),研究了grna對(duì)肝癌hepg-2細(xì)胞的影響。細(xì)胞劃痕實(shí)驗(yàn)結(jié)果證實(shí)grna處理可以顯著抑制hepg-2細(xì)胞的遷移作用,用2μmgrna處理hepg-2細(xì)胞12、24h,腫瘤細(xì)胞的遷移率分別為29.60±2.33%和40.97±1.23%,顯著低于對(duì)照組。transwell實(shí)驗(yàn)進(jìn)一步證實(shí)grna對(duì)hepg-2細(xì)胞遷移具有顯著的抑制作用,用0、0.5、1.0、2.0μmgrna分別處理hepg-2細(xì)胞48h后,遷移到transwell小室下層的數(shù)目由378±28分別減少到297±35、210±13、146±17。enoblock處理也呈現(xiàn)類似的結(jié)果。另外,grna可以顯著抑制hepg-2細(xì)胞的侵襲作用,且呈劑量依賴性。0、0.5、1.0、2.0μm濃度的grna處理hepg-2細(xì)胞后,小室下層的數(shù)目由208±16減少到167±16、115±3、62±9,這種作用與enoblock處理后對(duì)hepg-2細(xì)胞侵襲的抑制作用類似。上述結(jié)果說明,grna與eno1抑制劑enablock的作用一樣,可以抑制腫瘤細(xì)胞的遷移與侵襲。為了研究grna對(duì)腫瘤細(xì)胞功能的影響與eno1的相關(guān)性,我們研究了在eno1過表達(dá)條件下grna對(duì)肝癌細(xì)胞hepg2功能的影響。發(fā)現(xiàn)高表達(dá)eno1可以拮抗grna對(duì)hepg-2細(xì)胞遷移和侵襲的抑制作用。transwell實(shí)驗(yàn)結(jié)果顯示,過表達(dá)eno1可以增加肝癌hepg-2細(xì)胞的遷移;遷移細(xì)胞的數(shù)目從227±15增加到427±49,這說明eno1有促進(jìn)細(xì)胞遷移的能力。grna處理肝癌細(xì)胞后,細(xì)胞的遷移數(shù)由227±15降低至144±21,但在eno1高表達(dá)的肝癌細(xì)胞中,grna處理后,細(xì)胞遷移數(shù)從144±21增加到346±46。同樣,過表達(dá)eno1可以拮抗grna對(duì)hepg-2細(xì)胞侵襲的抑制作用,在eno1高表達(dá)的肝癌細(xì)胞中,hepg-2侵襲數(shù)目從67±10增加到112±16。以上實(shí)驗(yàn)結(jié)果表明grna可以與eno1結(jié)合,并通過影響eno1的功能抑制腫瘤細(xì)胞的遷移和侵襲。為了研究grna對(duì)凋亡和糖異生相關(guān)蛋白表達(dá)的影響和eno1的相關(guān)性,我們研究了eno1過表?xiàng)l件下grna對(duì)凋亡和糖異生相關(guān)蛋白表達(dá)的影響。我們發(fā)現(xiàn),grna處理過后的細(xì)胞抗凋亡蛋白akt、bcl-xl、c-myc蛋白明顯減少;過表達(dá)eno1后蛋白濃度明顯增加。而相同濃度grna處理hepg-2細(xì)胞后,過表達(dá)eno1的細(xì)胞中的抗凋亡蛋白濃度明顯高于非過表達(dá)eno1細(xì)胞,這說明grna是通過與eno1結(jié)合作用誘導(dǎo)細(xì)胞凋亡。相似地,grna可以通過作用eno1調(diào)控pck1、pck2影響hepg-2細(xì)胞葡萄糖攝取。以上實(shí)驗(yàn)結(jié)果證明grna可以與eno1結(jié)合,并通過影響eno1的功能影響相關(guān)抗凋亡蛋白和糖異生蛋白的表達(dá)。綜上所述,我們的研究結(jié)果證實(shí),grna可以誘導(dǎo)腫瘤細(xì)胞凋亡、抑制腫瘤細(xì)胞的遷移與侵襲,增加腫瘤細(xì)胞葡萄糖的攝取。grna可以與eno1發(fā)生特異性相互作用,eno1過表達(dá)可以促進(jìn)腫瘤細(xì)胞的遷移與侵襲,并能拮抗grna對(duì)腫瘤細(xì)胞凋亡、遷移與侵襲及葡萄糖攝取的功能的影響。這說明grna與eno1的相互作用是影響腫瘤細(xì)胞功能的重要因素。本研究對(duì)全面認(rèn)識(shí)ENO1的生物學(xué)功能具有重要價(jià)值,對(duì)發(fā)展ENO1作為抗腫瘤藥物靶標(biāo)也具有重要意義。
[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.

【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96

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本文編號(hào)：1767162