硒結(jié)合蛋白1生物功能及抗腫瘤作用機理的研究
發(fā)布時間：2018-09-06 10:50
【摘要】：硒做為一種人體必須的微量元素對于人體各種機能的提高和多種疾病的預防和治療都具有潛在的重要價值,長期較低的硒攝入量對人體健康具有多種不利的影響。由于我國約有70%的土地處在全球的缺硒帶上,其上產(chǎn)出的各種動、植物和微生物產(chǎn)品都屬于缺硒產(chǎn)品,而無機碼的攝入又會對人體產(chǎn)生多種毒副作用,因此硒蛋白做為一種有機硒的代表成為各種富硒食品和硒相關(guān)藥品研究的熱點。然而硒蛋白的生物功能多樣,作用靶點廣泛,具體抗病機制尚不清楚,阻礙了其在醫(yī)藥和食品中的進一步應用,因此對硒蛋白結(jié)構(gòu)和功能以及抗病分子機制的研究具有重要意義。硒結(jié)合蛋白1(SBP1)做為一種新型的含硒蛋白可能參與了人體細胞高爾基體蛋白運輸?shù)榷鄠�重要的生化進程,并通過與多種其它蛋白的相互作用影響細胞內(nèi)的信號傳導發(fā)揮其潛在的抑癌功能。本研究旨在通過蛋白組學的方法對SBP1的生物功能進行研究揭示其參與癌癥發(fā)生的具體信號通路和分子機制,并通過SBP1與谷胱甘肽過氧化物酶(GPx1)的相互作用研究以及兩者與硒的協(xié)同作用揭示新的腫瘤發(fā)病機制,進一步了解SBP1的結(jié)構(gòu),并為硒所介導的個性化腫瘤預防提供重要的理論基礎。主要研究結(jié)果分述如下:1.研究了SBP1在體內(nèi)和體外對腫瘤細胞生物學行為的影響利用誘導型病毒載體pRetroX-Tight-Pur構(gòu)建了SBP1哺乳動物細胞誘導質(zhì)粒pRetroX-Tight-Pur-SBP1,并通過單克隆篩選得到了SBP1穩(wěn)轉(zhuǎn)誘導結(jié)腸癌細胞HCT116-TetSBP1。使用該細胞在體外培養(yǎng)條件下大量誘導SBP1蛋白的表達,并測定腫瘤細胞各種生物學行為的變化。通過對細胞增殖,細胞遷移和細胞凋亡的檢測發(fā)現(xiàn)SBP1的誘導表達對體外培養(yǎng)腫瘤細胞的增殖和遷移影響不大,能夠誘導部分細胞發(fā)生凋亡但效果不顯著。將誘導SBP1表達的HCT116-TetSBP1細胞注射到裸鼠體內(nèi),并在體內(nèi)維持SBP1的誘導表達,檢測其對腫瘤細胞生長和轉(zhuǎn)移的影響,發(fā)現(xiàn)體內(nèi)誘導SBP1表達不僅顯著抑制了腫瘤細胞在裸鼠皮下的生長而且顯著減少了腫瘤細胞向肺部的轉(zhuǎn)移。2.利用iTRAQ蛋白組學的方法研究了SBP1的體內(nèi)抑癌功能從裸鼠皮下種植瘤形成的腫瘤組織中提取蛋白,使用iTRAQ蛋白組學的方法鑒定差異表達的蛋白,結(jié)果共鑒定到1975個蛋白,其中差異顯著蛋白132個(p0.05)。通過生物信息學GO生物功能分析和KEGG信號通路分析,篩選到了13個細胞脂代謝相關(guān)蛋白,其中5個為表達上調(diào)蛋白包括DKK1,HSP60,DHCR7,ANXA4和AGPAT5;8個為表達下調(diào)蛋白包括LPCAT2,GPx5,GAPDH,NME2,ALDH2,BPIFA3,PPIA和FABP4。另外篩選到7個細胞糖代謝相關(guān)蛋白包括GAA,GAPDH,ALDH2,TXN,EN03,UGDH和LUM,全部為表達下調(diào)蛋白。對iTRAQ實驗結(jié)果進行了Western blotting驗證,顯示iTRAQ分析結(jié)果準確可靠。通過檢測多種信號通路蛋白表達的變化,表明SBP1對細胞脂代謝和糖代謝以及癌癥相關(guān)的多重信號通路都有影響。由此推測SBP1通過影響細胞糖代謝和脂代謝相關(guān)蛋白的表達參與了細胞內(nèi)多個信號通路的信號傳導,包括EMT通路、WNT信號通路、JNK信號通路、NOTCH信號通路和NFkB信號通路,并通過這些癌癥相關(guān)信號通路引起一系列下游蛋白的激活或抑制,從而發(fā)揮其抑癌功能。3.研究了SBP1硒結(jié)合位點對其功能的影響對SBP1硒結(jié)合位點第57位氨基酸進行了單核苷酸突變,使其密碼子由TGC突變?yōu)镚GC,編碼的氨基酸由半胱氨酸(Cys,C)突變?yōu)楦拾彼?Gly,G),去除了原有氨基酸上的一個巰基,使得SBP1失去了結(jié)合硒的能力,而對其蛋白質(zhì)結(jié)構(gòu)不會產(chǎn)生太大的影響。構(gòu)建了突變SBP1哺乳動物細胞表達質(zhì)粒pIRES2-SBP1C57G,將其轉(zhuǎn)入人結(jié)腸癌細胞HCT116檢測對SBP1功能的影響。結(jié)果顯示:(1)硒造成的細胞毒性在有SBP1表達的細胞中導致的死亡率為38.2%,而在SBP1C57G表達的細胞中導致的死亡率為64.4%,因此SBP1C57G的點突變顯著降低了SBP1對細胞的保護作用;(2)通過不同MCF7細胞中SBP1半衰期的檢測,表明兩種不同多態(tài)性的GPx1蛋白(GPx1A5P和GPx1A7L)的存在都會將SBP1蛋白的半衰期從45h延長到60h左右,而SBP1C57G的半衰期在GPx1存在時依然會從60h減少到45h,表明SBP1硒結(jié)合位點的突變破壞了這兩種蛋白之間的結(jié)合;(3)通過多個信號通路蛋白和細胞內(nèi)ATP含量的檢測,表明SBP1C57G引起了細胞ATP總含量的降低,進而導致不依賴于信號通路的多個蛋白磷酸化的減少。4.研究了SBP1和GPx1兩種蛋白在體外的相互作用構(gòu)建了大腸桿菌重組表達載體PQE80L-SBP1和pQE80L-GPx1,并利用大腸桿菌表達宿主SoluBL21實現(xiàn)了SBP1的可溶性表達。通過鎳柱純化和分子篩純化方法分別得到了電泳純的SBP1蛋白和GPx1蛋白。利用兩種純化蛋白在體外進行相互作用后,發(fā)現(xiàn)經(jīng)過Superdex 200凝膠層析的洗脫時間從原有的34 min(SBP1)和30 min(GPx1)分別延長至42.5 min和37 min,可能是由于兩種蛋白之間形成了多蛋白復合體所造成的。從人結(jié)腸癌細胞HCT116中獲取與SBP1和GPx1相互作用蛋白的研究表明,兩種蛋白具有相同的作用蛋白硒代半胱氨酸特異延長因子(EEFSEC)。
[Abstract]:Selenium, as a necessary trace element for human body, has potential important value for the improvement of human function and the prevention and treatment of various diseases. Long-term low selenium intake has many adverse effects on human health. Selenoprotein, as a representative of organic selenium, has become a research hotspot in various selenium-rich foods and selenium-related drugs. However, selenoprotein has a variety of biological functions, a wide range of targets, and the specific mechanism of disease resistance is still unclear. Selenium binding protein 1 (SBP1), as a new selenium-containing protein, may be involved in many important biochemical processes such as Golgi protein transport in human cells and may be involved in many other proteins. The purpose of this study is to reveal the specific signaling pathways and molecular mechanisms involved in the development of cancer by proteomics methods, and to study the interaction between SBP1 and glutathione peroxidase (GPx1) and the biological functions of SBP1. The synergistic effects of SBP1 and selenium reveal new mechanisms of tumor pathogenesis, further understand the structure of SBP1, and provide an important theoretical basis for selenium-mediated personalized tumor prevention. The main results are summarized as follows: 1. The effects of SBP1 on the biological behavior of tumor cells in vitro and in vivo were studied using inducible virus vector pRetroX-Tight-Pur. SBP1 mammalian cell-induced plasmid pRetroX-Tight-Pur-SBP1 was constructed, and the stable transfection of SBP1 into colon cancer cell HCT116-TetSBP1 was obtained by monoclonal screening. The expression of SBP1 protein was induced by SBP1 cells in vitro and the changes of biological behavior of tumor cells were measured. Migration and apoptosis detection showed that the induction of SBP1 had little effect on the proliferation and migration of tumor cells in vitro, and could induce apoptosis of some cells, but the effect was not significant. In vivo, the expression of SBP1 not only significantly inhibited the growth of tumor cells subcutaneously in nude mice, but also significantly reduced the metastasis of tumor cells to the lungs. 2. Using iTRAQ proteomics method, the antitumor function of SBP1 in vivo was studied. Proteins were extracted from tumor tissues formed by subcutaneous implantation of tumor in nude mice and iTRAQ eggs were used. The results showed that 132 of the 1975 proteins were significantly different (p0.05). Through bioinformatics GO functional analysis and KEGG signaling pathway analysis, 13 proteins related to lipid metabolism were screened. Five of them were up-regulated proteins, including DKK1, HSP60, DHCR7, ANXA4 and AGPAT5. Seven proteins related to glucose metabolism, including GAA, GAPDH, ALDH2, BPIFA3, PPIA and FABP4, were screened out, including GAA, GAPDH, ALDH2, TXN, EN03, UGDH and LUM. All of them were down-regulated proteins. Western blotting was used to verify the results of iTRAQ assay. The results showed that iTRAQ analysis was accurate and reliable. The changes of protein expression of SBP1 signaling pathway indicate that SBP1 has effects on cell lipid metabolism, glucose metabolism and cancer-related multiple signaling pathways. The pathway, NOTCH signaling pathway and NFkB signaling pathway can activate or inhibit a series of downstream proteins through these cancer-related signaling pathways, thus exerting their anticancer function. 3. The effect of SBP1 selenium binding site on its function was studied. The single nucleotide mutation of amino acid at position 57 of SBP1 selenium binding site was carried out to make its codon protrude from TGC. The mutant SBP1 mammalian cell expression plasmid pIRES2-SBP1C57G was constructed and transformed into human colon cancer. The results showed that: (1) Selenium-induced cytotoxicity resulted in a mortality rate of 38.2% in SBP1-expressing cells and 64.4% in SBP1C57G-expressing cells, so point mutation of SBP1C57G significantly reduced the protective effect of SBP1 on SBP1 cells; (2) SBP1 in different MCF7 cells was induced by point mutation of SBP1C57G. Half-life test showed that the existence of two different polymorphisms of GPx1 protein (GPx1A5P and GPx1A7L) would prolong the half-life of SBP1 protein from 45 hours to about 60 hours, while the half-life of SBP1C57G would still be reduced from 60 hours to 45 hours in the presence of GPx1, suggesting that the mutation of SBP1 selenium binding site destroyed the binding between the two proteins; (3) through multiple Detection of signal pathway proteins and intracellular ATP levels showed that SBP1C57G caused a decrease in total ATP content in cells, which led to a decrease in phosphorylation of multiple proteins independent of signal pathways. 4. The interaction between SBP1 and GPx1 proteins in vitro was studied to construct recombinant expression vectors PQE80L-SBP1 and pQE80L-GPx1, and the recombinant expression vectors PQE80L-GPx1 were constructed. Soluble expression of SBP1 was achieved by E.coli expression host SoluBL21. Electrophoretic purified SBP1 and GPx1 proteins were obtained by nickel column purification and molecular sieve purification, respectively. After interaction between the two purified proteins in vitro, it was found that the elution time was 34 min (SBP1) and 30 min (SBP1) after Superdex 200 gel chromatography. (GPx1) was prolonged to 42.5 min and 37 min respectively, possibly due to the formation of multiple protein complexes between the two proteins. Studies on the protein interacting with SBP1 and GPx1 obtained from human colon cancer cell HCT116 showed that the two proteins had the same function protein, selenocysteine specific elongation factor (EEFSEC).
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R73-3
本文編號：2226126
[Abstract]:Selenium, as a necessary trace element for human body, has potential important value for the improvement of human function and the prevention and treatment of various diseases. Long-term low selenium intake has many adverse effects on human health. Selenoprotein, as a representative of organic selenium, has become a research hotspot in various selenium-rich foods and selenium-related drugs. However, selenoprotein has a variety of biological functions, a wide range of targets, and the specific mechanism of disease resistance is still unclear. Selenium binding protein 1 (SBP1), as a new selenium-containing protein, may be involved in many important biochemical processes such as Golgi protein transport in human cells and may be involved in many other proteins. The purpose of this study is to reveal the specific signaling pathways and molecular mechanisms involved in the development of cancer by proteomics methods, and to study the interaction between SBP1 and glutathione peroxidase (GPx1) and the biological functions of SBP1. The synergistic effects of SBP1 and selenium reveal new mechanisms of tumor pathogenesis, further understand the structure of SBP1, and provide an important theoretical basis for selenium-mediated personalized tumor prevention. The main results are summarized as follows: 1. The effects of SBP1 on the biological behavior of tumor cells in vitro and in vivo were studied using inducible virus vector pRetroX-Tight-Pur. SBP1 mammalian cell-induced plasmid pRetroX-Tight-Pur-SBP1 was constructed, and the stable transfection of SBP1 into colon cancer cell HCT116-TetSBP1 was obtained by monoclonal screening. The expression of SBP1 protein was induced by SBP1 cells in vitro and the changes of biological behavior of tumor cells were measured. Migration and apoptosis detection showed that the induction of SBP1 had little effect on the proliferation and migration of tumor cells in vitro, and could induce apoptosis of some cells, but the effect was not significant. In vivo, the expression of SBP1 not only significantly inhibited the growth of tumor cells subcutaneously in nude mice, but also significantly reduced the metastasis of tumor cells to the lungs. 2. Using iTRAQ proteomics method, the antitumor function of SBP1 in vivo was studied. Proteins were extracted from tumor tissues formed by subcutaneous implantation of tumor in nude mice and iTRAQ eggs were used. The results showed that 132 of the 1975 proteins were significantly different (p0.05). Through bioinformatics GO functional analysis and KEGG signaling pathway analysis, 13 proteins related to lipid metabolism were screened. Five of them were up-regulated proteins, including DKK1, HSP60, DHCR7, ANXA4 and AGPAT5. Seven proteins related to glucose metabolism, including GAA, GAPDH, ALDH2, BPIFA3, PPIA and FABP4, were screened out, including GAA, GAPDH, ALDH2, TXN, EN03, UGDH and LUM. All of them were down-regulated proteins. Western blotting was used to verify the results of iTRAQ assay. The results showed that iTRAQ analysis was accurate and reliable. The changes of protein expression of SBP1 signaling pathway indicate that SBP1 has effects on cell lipid metabolism, glucose metabolism and cancer-related multiple signaling pathways. The pathway, NOTCH signaling pathway and NFkB signaling pathway can activate or inhibit a series of downstream proteins through these cancer-related signaling pathways, thus exerting their anticancer function. 3. The effect of SBP1 selenium binding site on its function was studied. The single nucleotide mutation of amino acid at position 57 of SBP1 selenium binding site was carried out to make its codon protrude from TGC. The mutant SBP1 mammalian cell expression plasmid pIRES2-SBP1C57G was constructed and transformed into human colon cancer. The results showed that: (1) Selenium-induced cytotoxicity resulted in a mortality rate of 38.2% in SBP1-expressing cells and 64.4% in SBP1C57G-expressing cells, so point mutation of SBP1C57G significantly reduced the protective effect of SBP1 on SBP1 cells; (2) SBP1 in different MCF7 cells was induced by point mutation of SBP1C57G. Half-life test showed that the existence of two different polymorphisms of GPx1 protein (GPx1A5P and GPx1A7L) would prolong the half-life of SBP1 protein from 45 hours to about 60 hours, while the half-life of SBP1C57G would still be reduced from 60 hours to 45 hours in the presence of GPx1, suggesting that the mutation of SBP1 selenium binding site destroyed the binding between the two proteins; (3) through multiple Detection of signal pathway proteins and intracellular ATP levels showed that SBP1C57G caused a decrease in total ATP content in cells, which led to a decrease in phosphorylation of multiple proteins independent of signal pathways. 4. The interaction between SBP1 and GPx1 proteins in vitro was studied to construct recombinant expression vectors PQE80L-SBP1 and pQE80L-GPx1, and the recombinant expression vectors PQE80L-GPx1 were constructed. Soluble expression of SBP1 was achieved by E.coli expression host SoluBL21. Electrophoretic purified SBP1 and GPx1 proteins were obtained by nickel column purification and molecular sieve purification, respectively. After interaction between the two purified proteins in vitro, it was found that the elution time was 34 min (SBP1) and 30 min (SBP1) after Superdex 200 gel chromatography. (GPx1) was prolonged to 42.5 min and 37 min respectively, possibly due to the formation of multiple protein complexes between the two proteins. Studies on the protein interacting with SBP1 and GPx1 obtained from human colon cancer cell HCT116 showed that the two proteins had the same function protein, selenocysteine specific elongation factor (EEFSEC).
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R73-3
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